Identification of polynucleotides and polypetide for predicting activity of compounds that interact with protein tyrosine kinase and or protein tyrosine kinase pathways

ABSTRACT

The present invention describes polynucleotides and polypeptides that have been discovered to correlate to the relative intrinsic sensitivity or resistance of cells, e.g., colon cell lines, to treatment with compounds that interact with and inhibit src tyrosine kinases. These polynucleotides and polypeptides have been shown, through a weighted voting cross validation program, to have utility in predicting the intrinsic resistance and sensitivity of colon cell lines to these compounds. Such polynucleotides and polypeptides whose expression levels correlate highly with drug sensitivity or resistance comprise predictor or marker sets of polynucleotides and polypeptides that are useful in methods of predicting drug response, and as prognostic or diagnostic indicators in disease management, particularly in those disease areas in which signaling through src tyrosine kinase of the src tyrosine kinase pathway is involved with the disease process.

This application claims benefit to provisional application U.S. Ser. No.60/350,061 filed Jan. 18, 2002. The entire teachings of the referencedapplication are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field ofpharmacogenomics, and more specifically to new and alternative methodsand procedures to determine drug sensitivity in patients to allow thedevelopment of individualized genetic profiles which aid in treatingdiseases and disorders based on patient response at a molecular level.

BACKGROUND OF THE INVENTION

The major goal of pharmacogenomics research is to identify geneticmarkers that accurately predict a given patient's response to drugs inthe clinic; such individualized genetic assessment would greatlyfacilitate personalized treatment. An approach of this nature isparticularly needed in cancer treatment and therapy, where commonly usedagents are ineffective in many patients, and side effects are frequent.

The classification of patient samples is a crucial aspect of cancerdiagnosis and treatment. The association of a patient's response to drugtreatment with molecular and genetic markers can open up newopportunities for drug development in non-responding patients, ordistinguish a drug's indication among other treatment choices because ofhigher confidence in the efficacy. Further, the pre-selection ofpatients who are likely to respond well to a medicine, drug, orcombination therapy may reduce the number of patients needed in aclinical study or accelerate the time needed to complete a clinicaldevelopment program (M. Cockett et al., 2000, Current Opinion inBiotechnology, 11:602-609).

The ability to predict drug sensitivity in patients is particularlychallenging because drug responses reflect not only properties intrinsicto the target cells, but also a host's metabolic properties. Efforts bythose in the art to use genetic information to predict drug sensitivityhave primarily focused on individual polynucleotides and polypeptidesthat have broad effects, such as the multidrug resistant polynucleotidesand polypeptides, mdr1 and mrp1 (P. Sonneveld, 2000, J. Intern. Med.,247:521-534). Microarray technologies have also made it morestraightforward to monitor simultaneously the expression pattern ofthousands of polynucleotides and polypeptides, to analyze multiplemarkers and to categorize cancers into subgroups (J. Khan et al., 1998,Cancer Res., 58:5009-5013; A. A. Alizadeh et al., 2000, Nature,403:503-511; M. Bittner et al., 2000, Nature, 406:536-540; J. Khan etal., 2001, Nature Medicine, 7(6):673-679; and T. R. Golub et al., 1999,Science, 286:531-537).

Such technologies and molecular tools have made it possible to monitorthe expression level of a large number of transcripts within a cell atany one time (see, e.g., Schena et al., 1995, Quantitative monitoring ofgene expression patterns with a complementary DNA micro-array, Science,270:467470; Lockhart et al., 1996, Expression monitoring byhybridization to high-density oligonucleotide arrays, NatureBiotechnology, 14:1675-1680; Blanchard et al., 1996, Sequence to array:Probing the genome's secrets, Nature Biotechnology, 14:1649; U.S. Pat.No. 5,569,588, issued Oct. 29, 1996 to Ashby et al.) In organisms,including humans, for which the complete genome is known, it is possibleto analyze the transcripts of all polynucleotides and polypeptideswithin the cell.

How differential gene expression is associated with health and diseaseis a basis of functional genomics, which is defined as the study of allof the polynucleotides and polypeptides expressed by a specific cell ora group of cells and the changes in their expression pattern duringdevelopment, disease, or environmental exposure. Hybridization arrays,used to study gene expression, allow gene expression analysis on agenomic scale by permitting the examination of changes in expression ofliterally thousands of polynucleotides and polypeptides at one time. Ingeneral, for hybridization arrays, gene-specific sequences (probes) areimmobilized on a solid state matrix. These sequences are then queriedwith labeled copies of nucleic acids from biological samples (targets).The underlying theory is that the greater the expression of a gene, thegreater the amount of labeled target and thus, the greater output ofsignal. (W. M. Freeman et al., 2000, BioTechniques), 29:1042-1055).

Recent studies have demonstrated that gene expression informationgenerated by microarray analysis of human tumors can predict clinicaloutcome (L. J. van't Veer et al., 2002, Nature, 415:530-536; M. West etal., 2001, Proc. Natl. Acad. Sci. USA, 98:11462-11467; T. Sorlie et al.,2001, Proc. Natl. Acad. Sci. USA, 98:10869-10874; M. Shipp et al., 2002,Nature Medicine, 8(1):68-74). These findings bring hope that cancertreatment will be vastly improved by better predicting the response ofindividual tumors to therapy.

Needed in the art are new and alternative methods and procedures todetermine drug sensitivity in patients to allow the development ofindividualized genetic profiles which aid in treating diseases anddisorders based on patient response at a molecular level. By usingcultured cells as a model of in vivo effects, the present inventionadvantageously focuses on cell-intrinsic properties that are exposed incell culture and involves identified polynucleotides and polypeptidesthat correlate with drug sensitivity. The presently described discoveryand identification of polynucleotides and polypeptides/markerpolynucleotides and polypeptides (predictor polynucleotides, predictorpolypeptides, predicter polynucleotide subsets, and predictorpolypeptide subsets) in cell lines assayed in vitro can be used tocorrelate with drug responses in vivo, and thus can be extended toclinical situations in which the same polynucleotides and polypeptidesare used to predict responses to drugs and/or chemotherapeutic agents bypatients.

SUMMARY OF THE INVENTION

The present invention describes the identification of markerpolynucleotides and polypeptides whose expression levels are highlycorrelated with drug sensitivity in colon cell lines that are eithersensitive or resistant to protein tyrosine kinase inhibitor compounds.More particularly, the protein tyrosine kinases that are inhibited inaccordance with the present invention include members of the Src familyof tyrosine kinases, for example, Src, Fgr, Fyn, Yes, Bik, Hck, Lck andLyn, as well as other protein tyrosine kinases, including, Bcr-abl, Jak,PDGFR, c-kit and Ephr. For a review of these and other protein tyrosinekinases, see, for example, P. Blume-Jensen and T. Hunter, 2001,“Oncogene Kinase Signaling”, Nature, 411:355-365. Some of thesepolynucleotides and polypeptides are also modulated by the tyrosinekinase inhibitor compounds, in particular, src tyrosine kinase inhibitorcompounds, which indicates their involvement in the protein tyrosinekinase signaling pathway. These polynucleotides and polypeptides or“markers” show utility in predicting a host's response to a drug and/ordrug treatment.

It is an object of this invention to provide a cell culture model toidentify polynucleotides and polypeptides whose expression levelscorrelate with drug sensitivity of cells associated with a diseasestate, or a host having a disease. In accordance with the presentinvention, oligonucleotide microarrays were utilized to measure theexpression levels of a large number of polynucleotides and polypeptidesin a panel of untreated cell lines, particularly colon cell lines, forwhich drug sensitivity to four src kinase inhibitor compounds wasdetermined. The determination of the gene expression profiles in thepreviously untreated cells allowed a prediction of chemosensitivity andthe identification of marker polynucleotides and polypeptides whoseexpression levels highly correlate with sensitivity to drugs orcompounds that modulate, preferably inhibit, src kinase or src familykinases or the pathway in which src or src family tyrosine kinases areinvolved. The marker or predictor polynucleotides and polypeptides arethus useful for predicting a patient's response to drugs or drugtreatments that directly or indirectly affect src or src family tyrosinekinases activity.

It is another object of the present invention to provide a method ofdetermining or predicting if an individual requiring drug orchemotherapeutic treatment or therapy for a disease state, for example,colon disease, or a cancer or tumor of a particular type, preferably, acolon cancer or tumor, will successfully respond or will not respond tothe drug or chemotherapeutic treatment or therapy, preferably atreatment or therapy involving a src or src family tyrosine kinasesmodulating agent, e.g., an inhibitor of src kinase activity, prior tosubjecting the individual to such treatment or chemotherapy. Preferably,the treatment or therapy involves a protein tyrosine kinase modulatingagent, e.g., an inhibitor of the protein tyrosine kinase activity. Theprotein tyrosine kinases whose activities can be inhibited by inhibitorcompounds according to this invention include, for example, members ofthe Src family of tyrosine kinases, for example, Src, Fgr, Fyn, Yes,Blk, Hck, Lck and Lyn, as well as other protein tyrosine kinases,including, Bcr-abl, Jak, PDGFR, c-kit and Ephr. In accordance with thepresent invention, cells from a patient tissue sample, e.g., a tumor orcancer biopsy, preferably a colon cancer or tumor sample, or sloughedcolonocytes, are assayed to determine their gene expression patternprior to treatment with a src or src family tyrosine kinases modulatingcompound or drug, preferably a src kinase inhibitor. The resulting geneexpression profile of the test cells before exposure to the compound ordrug is compared with the gene expression pattern of the predictor setof polynucleotides and polypeptides that have been described and shownherein (Tables 3-6) in the control panel of the untreated cells that areeither resistant or sensitive to the drug or compound, i.e., FIGS. 1-3.

In addition, in such a method, the gene expression pattern of subsets ofpredictor polynucleotides and polypeptides, comprising at least about 5,at least about 10, at least about 15, at least about 20, at least about25, at least about 30, at least about 40, at least about 45 at leastabout 50, or more, polynucleotides and polypeptides may be used. In thiscontext, the term “about” may be construed to mean 1, 2, 3, 4, or 5 moreor less polynucleotides or polypeptides within each predicter subset.Preferably, in such a method, the gene expression pattern of subsets ofpredictor polynucleotides and polypeptides, comprising sets of 25, 15and 10 polynucleotides and polypeptides as set forth in Tables 10 thru12, respectively, can also be used. These polynucleotides andpolypeptides are derived from the control panel of the untreated cellsthat have been determined to be either resistant or sensitive to thedrug or compound as shown herein.

Success or failure of treatment with a drug can be determined based onthe gene expression pattern of the test cells from the test tissue,e.g., tumor or cancer biopsy, as being relatively the same as ordifferent from the gene expression pattern of the predictor set ofpolynucleotides and polypeptides in the resistant or sensitive controlpanel of cells for which drug sensitivity to the src kinase inhibitorcompounds has been determined. Thus, if the test cells show a geneexpression profile which corresponds to that of the predictor set ofpolynucleotides and polypeptides in the control panel of cells which aresensitive to the drug or compound, it is highly likely or predicted thatthe individual's cancer or tumor will respond favorably to treatmentwith the drug or compound. By contrast, if the test cells show a geneexpression pattern corresponding to that of the predictor set ofpolynucleotides and polypeptides of the control panel of cells which areresistant to the drug or compound, it is highly likely or predicted thatthe individual's cancer or tumor will not respond to treatment with thedrug or compound.

It is an aspect of this invention to provide screening assays fordetermining if a cancer patient will be susceptible or resistant totreatment with a drug or compound, particularly, a drug or compounddirectly or indirectly involved in a protein tyrosine kinase activity ora protein tyrosine kinase pathway. Such protein tyrosine kinasesinclude, without limitation, members of the Src family of tyrosinekinases, for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as wellas other protein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kitand Ephr.

It is a further object of the present invention to provide screeningassays for determining if a patient's cancer tumor will be susceptibleor resistant to treatment with a drug or compound, particularly, a drugor compound directly or indirectly involved in src or src familytyrosine kinases activity or the src or src family tyrosine kinasespathway.

It is another object of the present invention to provide a method ofmonitoring the treatment of a patient having a disease treatable by acompound or agent that modulates a src tyrosine kinase by comparing theresistance or sensitivity gene expression profile of cells from apatient tissue sample, e.g., a tumor or cancer biopsy, preferably acolon cancer or tumor sample, prior to treatment with a drug or compoundthat inhibits src or src family tyrosine kinases activity and againfollowing treatment with the drug or compound. The isolated cells fromthe patient are assayed to determine their gene expression patternbefore and after exposure to a compound or drug, preferably a src kinaseinhibitor, to determine if a change of the gene expression profile hasoccurred so as to warrant treatment with another drug or agent, or todiscontinue current treatment. The resulting gene expression profile ofthe cells tested before and after treatment is compared with the geneexpression pattern of the predictor set of polynucleotides andpolypeptides that have been described and shown herein to be highlyexpressed in cells that are either resistant or sensitive to the drug orcompound.

Such a monitoring process can indicate success or failure of a patient'streatment with a drug or compound based on the gene expression patternof the cells isolated from the patient's sample, e.g., a tumor or cancerbiopsy, as being relatively the same as or different from the geneexpression pattern of the predictor gene set of the resistant orsensitive control panel of cells that have been exposed to the drug orcompound and assessed for their gene expression profilefollowing-exposure. Thus, if, after treatment with a drug or compound,the test cells show a change in their gene expression profile from thatseen prior to treatment to one which corresponds to that of the controlpanel of cells that are resistant to the drug or compound, it can serveas an indicator that the current treatment should be modified, changed,or even discontinued. Also, should a patient's response become one thatis sensitive to treatment by a src kinase inhibitor compound, based oncorrelation of the expression profile of the predictor polynucleotidesand polypeptides, the patient's treatment prognosis can be qualified asfavorable and treatment can continue. Such monitoring processes can berepeated as necessary or desired. The monitoring of a patient's responseto a given drug treatment can also involve testing the patient's cellsin the assay as described only after treatment, rather than before andafter treatment, with drug or active compound.

It is a further object of the present invention to provide predictorpolynucleotides and polypeptides and predictor sets of polynucleotidesand polypeptides as tools that have both diagnostic and prognostic valuein disease areas in which signaling through protein tyrosine kinase or aprotein tyrosine kinase pathway is of importance, e.g., in cancers andtumors, in immunological disorders, conditions or dysfunctions, or indisease states in which cell signaling and/or proliferation controls areabnormal or aberrant. Such protein tyrosine kinases whose direct orindirect modulation can be associated with a disease state or condition,include members of the Src family of tyrosine kinases, for example, Src,Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as other protein tyrosinekinases, including, Bcr-abl, Jak, PDGFR, c-kit and Ephr. In accordancewith this invention, the use of predictor polynucleotides andpolypeptides, or a predictor gene set, is to forecast or foretell anoutcome prior to having any knowledge about a biological system, or acellular response. Also according to this invention, the predictorpolynucleotides and polypeptides or predictor gene set is useful inpredicting the phenotype that is used to classify a biological system orresponse. For example, the classification of a cell line as “resistant”or “sensitive” is based on the log₁₀(IC₅₀) value of each cell line toone or more compounds (e.g., a src kinase inhibitor compound), relativeto the mean log₁₀(IC₅₀) value of a cell line panel (e.g., a thirty-onecolon cell line panel, as described herein) that has been previouslyexposed to the compounds and statistically assessed as to the expressionlevel of polynucleotides and polypeptides correlating to resistance orsensitivity following exposure to the one or more compounds.

It is yet another object of the present invention to providepolynucleotides and polypeptides, such as those listed in Tables 3-5, orthe common polynucleotides and polypeptides shown in Table 6 herein, toassemble predictor gene subsets such as in Tables 10-12 to be able topredict or reasonably foretell the likely effect of either src tyrosineinhibitor compounds or compounds that affect the src tyrosine kinasesignaling pathway in different biological systems, or for cellularresponses. The predictor gene sets can be used in in vitro assays ofdrug response by test cells to predict in vivo outcome. In accordancewith this invention, the various predictor gene sets described herein,or the combination of these predictor sets with other polynucleotidesand polypeptides or other co-variants of these polynucleotides andpolypeptides, can be used, for example, to predict how patients withcancer or a tumor might respond to therapeutic intervention withcompounds that modulate the src tyrosine kinase family. In addition,such predictor sets can be used to predict how patients might respond totherapeutic intervention(s) that modulate(s) signaling through theentire src tyrosine kinase regulatory pathway. The predictor sets ofpolynucleotides and polypeptides, or co-variants of thesepolynucleotides and polypeptides, can be used to predict how patientswith a cancer or tumor respond to therapy employing compounds thatmodulate a tyrosine kinase, or the activity of a tyrosine kinase, suchas protein tyrosine kinase members of the Src family, for example, Src,Fgr, Fyn, Yes, BLk, Hck, Lck and Lyn, as well as other protein tyrosinekinases, including, Bcr-abl, Jak, PDGFR, c-kit and Ephr.

A further object of the present invention is to provide polynucleotidesand polypeptides comprising one or more predictor sets ofpolynucleotides and polypeptides that most highly correlate withresistance or sensitivity to drugs or compounds which are directly orindirectly involved with modulation of src tyrosine kinase or srctyrosine kinase signaling pathways. In accordance with this invention,predictor gene sets associated with resistance or sensitivity to srctyrosine kinase inhibitor compounds comprise the polynucleotides andpolypeptides presented in FIGS. 1-3 and Tables 3-6 herein. Alsoaccording to the invention, the polynucleotides and polypeptides ofTables 3-6 have been discovered to be expressed by cells which aresensitive or resistant to four different src kinase inhibitor compounds.The expression of these polynucleotides and polypeptides, orcombinations thereof, has been found to be highly correlated withsensitivity of cells to the different src kinase inhibitors. Theexpression patterns of the three sets of polynucleotides andpolypeptides correlating with sensitivity of thirty-one colon cells tothe src kinase inhibitor compounds are provided in FIGS. 1-3.

Yet another object of the present invention is to provide predictorpolynucleotides and polypeptides or predictor gene sets having bothdiagnostic and prognostic value in disease areas in which signalingthrough src tyrosine kinase or the src tyrosine kinase pathway isinvolved, e.g., in cancers or tumors, or in disease states in which cellsignaling and/or cellular proliferation controls are abnormal oraberrant. Also provided by this invention are common polynucleotides andpolypeptides whose expression levels are strongly correlated with eithersensitivity or resistance to all four of the src kinase inhibitorcompounds (Table 6). Because these polynucleotides and polypeptidescorrelate to drug sensitivity and resistance classifications associatedwith all four of the src kinase inhibitor compounds in cells, suchpolynucleotides and polypeptides can be used to build predictors ormarkers for other biological systems in which src kinase activity or srcor src family tyrosine kinases signaling pathways are involved.

Another object of the present invention is to provide one or morespecialized microarrays, e.g., oligonucleotide microarrays or cDNAmicroarrays, comprising those polynucleotides and polypeptides, orcombinations thereof, as described herein showing expression profilesthat correlate with either sensitivity or resistance to one or more srckinase inhibitor compounds. Such microarrays can be employed in in vitroassays for assessing the expression level of the polynucleotides andpolypeptides on the microarrays in the test cells from tumor biopsies,for example, and determining whether these test cells will be likely tobe resistant or sensitive to src kinase inhibitor compounds. Forexample, one or more microarrays can be prepared using each of thepolynucleotides and polypeptides, or combinations thereof, as describedherein and shown in FIGS. 1-3 and Tables 3-6. Cells from a tissue ororgan biopsy can be isolated and exposed to one or more of the inhibitorcompounds.

Following application of nucleic acids isolated from both untreated andtreated cells to one or more of the specialized microarrays, the patternof gene expression of the tested cells can be determined and comparedwith that of the predictor gene pattern from the panel of cells used tocreate the predictor gene set on the microarray. Based upon the geneexpression pattern results from the cells undergoing testing, it can bedetermined if the cells show a resistant or a sensitive profile of geneexpression. Whether or not the tested cells from a tissue or organbiopsy will respond to one or more of the inhibitor compounds and thecourse of treatment or therapy can then be determined or evaluated basedon the information gleaned from the results of the specializedmicroarray analysis.

It is a further object of the present invention to provide a kit fordetermining or predicting drug susceptibility or resistance by a patienthaving a disease, with particular regard to a cancer or tumor, namely, acolon cancer or tumor. Such kits would be useful in a clinical settingfor use in testing patient's biopsied tumor or cancer samples, forexample, to determine or predict if the patient's tumor or cancer willbe resistant or sensitive to a given treatment or therapy with a drug,compound, chemotherapy agent, or biological agent that are directly orindirectly involved with modification, preferably, inhibition, of srctyrosine kinase activity or a cell signaling pathway involving srctyrosine kinase activity. Provided in the kit are one or more predictorgene sets, preferably comprising one or more microarrays, e.g.,oligonucleotide microarrays or cDNA microarrays, comprising thosepolynucleotides and polypeptides that correlate with resistance andsensitivity to Src family of protein tyrosine kinases, for example, Src,Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as inhibitors of theBcr-abl, Jak, PDGFR, c-kit and Ephr protein tyrosine kinases; and, insuitable containers, the modulator agents/compounds for use in testingcells from patient tissue specimens or patient samples forresistance/sensitivity to compounds that inhibit src or src familytyrosine kinases activity; and instructions for use. In addition, kitscontemplated by the present invention also include reagents or materialsfor the monitoring of the expression of the predictor or markerpolynucleotides and polypeptides of the invention at the level of mRNAor protein, using other techniques and systems practiced in the art,e.g., RT-PCR assays, which employ primers designed on the basis of oneor more of the predictor polynucleotides and polypeptides describedherein, immunoassays, such as enzyme linked immunosorbent assays(ELISAs), immunoblotting, e.g., Western blots, or in situ hybridization,and the like, as further described herein.

Another object of the present invention is to provide one or morepolynucleotides and polypeptides among those of the predictorpolynucleotides and polypeptides identified herein that can serve astargets for the development of drug therapies for disease treatment.Such targets may be particularly applicable to treatment of colondisease, such as colon cancers or tumors. Because these predictorpolynucleotides and polypeptides are differentially expressed insensitive and resistant cells, their expression pattern is correlatedwith the relative intrinsic sensitivity of cells to treatment withcompounds that interact with and/or inhibit protein tyrosine kinases,including members of the Src family of protein tyrosine kinases, forexample, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as theBcr-abl, Jak, PDGFR, c-kit and Ephr protein tyrosine kinases.Accordingly, the polynucleotides and polypeptides highly expressed inresistant cells can serve as targets for the development of new drugtherapies for those tumors which are resistant to protein tyrosinekinase inhibitor compounds.

Yet another object of the present invention is to provide antibodies,either polyclonal or monoclonal, directed against one or more of the srcbiomarker polypeptides, or peptides thereof, encoded by the predictorpolynucleotides and polypeptides. Such antibodies can be used in avariety of ways, for example, to purify, detect, and target the srcbiomarker polypeptides of the present invention, including both in vitroand in vivo diagnostic, detection, screening, and/or therapeuticmethods, and the like. Included among the protein tyrosine kinasebiomarker polypeptides of this invention are members of the Src familyof protein tyrosine kinases, for example, Src, Fgr, Fyn, Yes, Blk, Hck,Lck and Lyn, as well as the Bcr-abl, Jak, PDGFR, c-kit and Ephr proteintyrosine kinases.

Further objects, features, and advantages of the present invention willbe better understood upon a reading of the detailed description of theinvention when considered in connection with the accompanying figures ordrawings.

DESCRIPTION OF THE FIGURES

The file of this patent contains at least one Figure executed in color.Copies of this patent with color Figure(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 illustrates a gene expression pattern according to the presentinvention. The 123 polynucleotides and polypeptides that most highlycorrelated with a resistance/sensitivity phenotype classification of the31 colon cell lines for BMS-A or BMS-D are shown. Each row correspondsto a gene, with the columns corresponding to expression levels in thedifferent cell lines. Expression levels for each gene are normalizedacross the median expression level of all the 31 cell lines. Thepolynucleotides and polypeptides with expression levels greater than themedian are shaded in red, and those below the median are shaded ingreen. The individual polynucleotides and polypeptides encoding the srcbiomarkers of the FIG. 1 are in the order as listed in Table 3.

FIG. 2 illustrates a gene expression pattern according to the presentinvention. The 119 polynucleotides and polypeptides most highlycorrelated with a resistance/sensitivity phenotype classification of the31 colon cell lines for BMS-B are shown. Each row corresponds to a gene,with the columns corresponding to expression levels in the differentcell lines. Expression levels for each gene are normalized across themedian expression level of all the 31 cell lines. The polynucleotidesand polypeptides with expression levels greater than the median areshaded in red, and those below the median are shaded in green. Theindividual polynucleotides and polypeptides encoding the src biomarkersof the FIG. 2 are in the order as listed in Table 4.

FIG. 3 illustrates a gene expression pattern according to the presentinvention. The 137 polynucleotides and polypeptides most highlycorrelated with a resistance/sensitivity phenotype classification of the31 colon cell lines for BMS-C are shown. Each row corresponds to a gene,with the columns corresponding to expression levels in the differentcell lines. Expression levels for each gene are normalized across themedian expression level of all the 31 cell lines. The polynucleotidesand polypeptides with expression levels greater than the median areshaded in red, and those below the median are shaded in green. Theindividual polynucleotides and polypeptides encoding the src biomarkersof the FIG. 3 are in the order as listed in Table 5.

FIG. 4 shows the error rates of prediction for the four src kinaseinhibitor compounds, BMS-A, BMS-B, BMS-C and BMS-D in cross validationand random permutation tests. The Genecluster software was used toselect polynucleotides and polypeptides and predict classificationsusing a “weighted-voting ‘leave one out’ cross-validation algorithm”, asdescribed herein. A different number of polynucleotides and polypeptideswas used in the predictor set for predicting resistant and sensitiveclasses to BMS-A, BMS-B, BMS-C and BMS-D in the colon cell lines. Thereal error rates were compared with the real error rates using the samenumber of polynucleotides and polypeptides as the predictor set in 20cases, in which classification for the colon cell lines was randomlyassigned. For example, when each predictor set contained 20polynucleotides and polypeptides, the real error rate of prediction forBMS-A or BMS-D was 15.7%; for BMS-B and BMS-C, the real error rates were19% and 16.2%, respectively. These error rate values are significantlylower than the real error rates obtained when random phenotypeclassifications are used for the cell lines (i.e., in a range of from30% to 70%).

DESCRIPTION OF THE TABLES

Table 1 shows the mean IC₅₀ of four src kinase inhibitors for each ofthe thirty-one colon cell lines. Thirty-one colon cell lines weretreated with each of the four src tyrosine kinase inhibitor compounds,namely, BMS-A, BMS-B, BMS-C and BMS-D, and the IC₅₀ was assessed in thecells by MTS assays as described in Example 1 (Methods). The mean IC₅₀values along with standard deviations (SD) were calculated from 2 to 5individual determinations for each cell line for the results shown. TheIC₅₀ unit is μM.

Table 2 shows the resistance/sensitivity classification of 31 colon celllines for the four src kinase inhibitor compounds BMS-A, BMS-B, BMS-Cand BMS-D. For each compound, the IC₅₀ for each cell line waslog-transformed to log₁₀(IC₅₀), and the log₁₀(ICso) values were thennormalized to the mean log₁₀(IC₅₀) across the 31 colon cell lines. Thecell lines with log₁₀(IC₅₀) below the mean log₁₀(IC₅₀) of all 31 celllines were defined as sensitive to the compound, while those withlog₁₀(IC₅₀) above the mean log₁₀(IC₅₀) were considered to be resistant.

Table 3 shows a gene list that demonstrated a high correlation betweenexpression pattern and resistance/sensitivity classification to BMS-A orBMS-D. The gene number, relative expression pattern, i.e., sensitive orresistant, Gene Accession number, gene description (Unigene cluster),SEQ ID NO: for the DNA sequence of the gene, and SEQ ID NO: for theamino acid sequence of the gene (if available), are presented in thetable. For each gene, the DNA and encoded amino acid sequencesrepresented by SEQ ID NOs. in the table are described in the SequenceListing.

Table 4 presents a gene list that demonstrated high correlation betweenexpression pattern and resistance/sensitivity classification to BMS-B.The gene number, relative expression pattern, i.e., sensitive orresistant, Gene Accession number, gene description (unigene cluster),SEQ ID NO: for the DNA sequence of the gene, and SEQ ID NO: for theamino acid sequence of the gene (if available), are presented in thetable. For each gene, the DNA and encoded amino acid sequencesrepresented by SEQ ID NOs. in the table are described in the SequenceListing.

Table 5 presents a gene list that demonstrated high correlation betweenexpression pattern and resistance/sensitivity classification to BMS-C.The gene number, relative expression pattern, i.e., sensitive orresistant, Gene Accession number, gene description (unigene cluster),SEQ ID NO: for the DNA sequence of the gene, and SEQ ID NO: for theamino acid sequence of the gene (if available), are presented in thetable. For each gene, the DNA and encoded amino acid sequencesrepresented by SEQ ID NOs. in the table are described in the SequenceListing.

Table 6 presents a common gene list from Tables 3-5 showing the highestcorrelation between expression pattern and resistance/sensitivityclassification of the cells to the four src kinase inhibitor compoundsBMS-A/BMS-D, BMS-B and BMS-C. The gene description, accession number,DNA sequence, amino acid sequence (if available), and the correspondingnucleic acid and amino acid SEQ ID NOS are provided. The relativeexpression patterns of each gene i.e., sensitive or resistant, areindicated.

Table 7 presents a resistance/sensitivity prediction of the 31 coloncell lines for BMS-A or BMS-D, BMS-B and BMS-C using 10 markers as apredictor set shown in Table 10. The true class is assigned as in Table2, based on the IC₅₀ results. The predicted class is determined by usingthe optimal 10 polynucleotides and polypeptides as the predictor set topredict the resistance or sensitive class. “S” represents Sensitive; “R”represents Resistant. The confidence score refers to prediction strengthfor each prediction made on a cell line by the predictor set. Theconfidence score ranges from 0 to 1, i.e., corresponding from low tohigh confidence in making the prediction. The error predictions areindicated by an asterisk (*).

Table 8 shows a resistance/sensitivity prediction of the 31 colon celllines for or BMS-D, BMS-B and BMS-C using 15 markers as a predictor setshown in Table 11. The true class is assigned as in Table 2, based onthe IC₅₀ results. The predicted class is determined by using the optimal15 polynucleotides and polypeptides as the predictor set to predict theresistance or sensitive class. “S” represents Sensitive; “R” representsResistant. The confidence score refers to prediction strength for eachprediction made on a cell line by the predictor set. The confidencescore ranges from 0 to 1, i.e., corresponding from low to highconfidence in making the prediction. The error predictions are indicatedby an asterisk (*).

Table 9 presents a resistance/sensitivity prediction of the 31 coloncell lines for BMS-A or BMS-D, BMS-B and BMS-C using 25 markers as apredictor set shown in Table 12. The true class is assigned as in Table2, based on the IC₅₀ results. The predicted class is determined by usingthe optimal 25 polynucleotides and polypeptides as the predictor set topredict the resistance or sensitive class. “S” represents Sensitive; “R”represents Resistant. The confidence score refers to prediction strengthfor each prediction made on a cell line by the predictor set. Theconfidence score ranges from 0 to 1, i.e., corresponding from low tohigh confidence in making the prediction. The error predictions areindicated by an asterisk (*).

Table 10 lists the predictor set of 10 polynucleotides and polypeptidesused in prediction as shown in Table 7. These 10 polynucleotides andpolypeptides were elected from the 73 common (as shown in Table 6). GeneAccession number, gene description (Unigene cluster), and relativeexpression pattern, i.e., sensitive or resistant, for this 10-genepredictor subset, are indicated.

Table 11 lists the predictor set of 15 polynucleotides and polypeptidesused in prediction as shown in Table 8. These 15 polynucleotides andpolypeptides were selected from the 73 common (as shown in Table 6).Gene Accession number, gene description (Unigene cluster), and relativeexpression pattern, i.e., sensitive or resistant, for this 15-genepredictor subset, are indicated.

Table 12 lists the predictor set of 25 polynucleotides and polypeptidesused in prediction as shown in Table 9. These 25 polynucleotides andpolypeptides were selected from the 73 common (as shown in Table 6).Gene Accession number, gene description (Unigene cluster), and relativeexpression pattern, i.e., sensitive or resistant, for this 25-genepredictor subset, are indicated.

Table 13 show representative forward and reverse RT-PCR primers for eachof the Src biomarker polynucleotides and polypeptides of the presentinvention, as identified by SEQ ID NO and Accession No. in Tables 3-5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the identification of polynucleotidesand polypeptides that correlate with drug sensitivity or resistanceemploying cell lines that are previously untreated with drug todetermine sensitivity of the cells to a drug, compound, or biologicalagent. These polynucleotides and polypeptides, called marker orpredictor polynucleotides and polypeptides herein, can be employed forpredicting drug response. The marker polynucleotides and polypeptideshave been determined in an in vitro assay employing microarraytechnology to monitor simultaneously the expression pattern of thousandsof discrete polynucleotides and polypeptides in previously untreatedcells, whose sensitivity to compounds or drugs, in particular, compoundsthat inhibit protein tyrosine kinase or protein tyrosine kinaseactivity, particularly src or src family tyrosine kinases, is tested.The protein tyrosine kinases, or activities thereof, associated withresponse to a drug, compound, or biological agent include, for example,members of the Src family of protein tyrosine kinases, for example, Src,Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as the Bcr-abl, Jak,PDGFR, c-kit and Ephr protein tyrosine kinases. (See, e.g., P.Blume-Jensen and T. Hunter, 2001, “Oncogene Kinase Signaling”, Nature,411:355-365).

This assay has allowed the identification of the marker polynucleotidesand polypeptides, called src biomarkers herein, having expression levelsin the cells that are highly correlated with drug sensitivity exhibitedby the cells. Such marker polynucleotides and polypeptides serve asuseful molecular tools for predicting a response to drugs, compounds,biological agents, chemotherapeutic agents, and the like, preferablythose drugs and compounds, and the like, that affect protein tyrosinekinase activity, particularly src or src family tyrosine kinasesactivity, via direct or indirect inhibition or antagonism of proteintyrosine kinase function, particularly src or src family tyrosinekinases function or activity.

In its preferred aspect, the present invention describes polynucleotidesand polypeptides that correlate with sensitivity or resistance of coloncell lines to treatment with protein tyrosine kinase inhibitorcompounds, particularly src tyrosine kinase inhibitor compounds asdescribed herein. The exposure of thirty-one colon cell lines to each offour src kinase inhibitor compounds provided a predictor set ofpolynucleotides and polypeptides for each compound that were most highlycorrelated with a resistance or sensitivity classification of thethirty-one colon cell lines to the inhibitor compounds. (FIGS. 1-3 andTables 3-5). The src kinase inhibitor compounds utilized for identifyingthe gene predictor sets of this invention are described in WO 00/62778,published Oct. 26, 2000. Specifically, for the four src kinase inhibitorcompounds analyzed, namely, BMS-A, BMS-B, BMS-C and BMS-D, the drugsensitivity classification for the thirty-one colon cell lines was thesame for BMS-A and BMS-D; and 26 out of 31 colon cell lines have thesame sensitivity classifications for all four src kinase inhibitorcompounds as shown in the Table 2. One or more of these four compoundshas a potent inhibitory activity for a number of protein tyrosinekinases, for example, members of the Src family of protein tyrosinekinases, including Src, Fgr, Fyn, Yes, BIk, Hck, Lck and Lyn, as well asthe Bcr-abl, Jak, PDGFR, c-kit and Ephr protein tyrosine kinases.Although the predicter gene sets are most useful in predicting efficacyof one or more of these compounds for inhibiting Src kinase functionand/or activity specifically, the predicter gene sers are also usefulfor predicting the efficacy of these compounds for inhibiting proteintyrosine kinases, in general, an in particularly Src, Fgr, Fyn, Yes,Blk, Hck, Lck and Lyn, as well as the Bcr-abl, Jak, PDGFR, c-kit andEphr protein tyrosine kinases.

The expression of 123, 119 and 137 predictor polynucleotides andpolypeptides, was found to correlate with resistance/sensitivity of thecolon cell lines to BMS-A/BMS-D, BMS-B and BMS-C respectively. Commonpredictor polynucleotides and polypeptides were also determined forpredicting a resistance/sensitivity classification of cells to the srckinase inhibitors. The common polynucleotides and polypeptides showingthe highest correlation between their expression pattern and theresistance or sensitivity classification in the cell lines for the srckinase inhibitor compounds are presented in Table 6.

In accordance with the invention, an approach has been discovered inwhich polynucleotides and polypeptides and combinations ofpolynucleotides and polypeptides have been identified whose expressionpattern, in a subset of cell lines, correlates to and can be used as anin vitro predictor of cellular response to treatment or therapy with onecompound, or with a combination or series of compounds, that are knownto inhibit or activate the function of a protein, enzyme, or molecule(e.g., a receptor) that is directly or indirectly involved in cellproliferation, cell responses to external stimuli, (such as ligandbinding), or signal transduction, e.g., a tyrosine kinase. Preferred areantagonists or inhibitors of the function of a given protein, e.g., atyrosine kinase.

In a preferred aspect, specific src tyrosine kinase inhibitor compounds,BMS-A, BMS-B, BMS-C and BMS-D were employed to determine drugsensitivity in a panel of colon cell lines following exposure of thecells to the compounds. Some of the cell lines were determined to beresistant to treatment with the inhibitor compounds, while others weredetermined to be sensitive to the inhibitors (Tables 1 and 2). A subsetof the cell lines examined provided an expression pattern or profile ofpolynucleotides and polypeptides, and combinations of polynucleotidesand polypeptides, that correlated to and serve as a predictor of, aresponse by the cells to these inhibitor compounds, and to compoundshaving similar modes of action and/or structure. (FIGS. 1-3 and Tables7-12).

Such a predictor set of cellular gene expression patterns correlatingwith sensitivity or resistance of cells following exposure of the cellsto a drug, or a combination of drugs, provides a useful tool forscreening a tumor sample before treatment with the drug, or a similardrug, or drug combination. The screening technique allows a predictionof cells of a tumor sample exposed to a drug, or a combination of drugs,based on the gene expression results of the predictor set, as to whetheror not the tumor, and hence a patient harboring the tumor, will or willnot respond to treatment with the drug or drug combination.

In addition, the predictor polynucleotides and polypeptides or predictorgene set can also be utilized as described herein for monitoring theprogress of disease treatment or therapy in those patients undergoingtreatment for a disease involving a src or src family tyrosine kinasesinhibitor compound or chemotherapeutic agent.

According to a particular embodiment of the present invention,oligonucleotide microarrays were utilized to measure the expressionlevels of over 12,000 polynucleotides and polypeptides in a panel ofthirty-one untreated colon cell lines for which the drug sensitivity tofour src kinase inhibitor compounds was determined. This analysis wasperformed to determine whether the gene expression signatures ofuntreated cells were sufficient for the prediction of chemosensitivity.Data analysis allowed the identification of marker polynucleotides andpolypeptides whose expression levels were found to be highly correlatedwith drug sensitivity. In addition, the treatment of untreated cellswith drug also provided gene expression signatures predictive ofresistance to the compounds. Subsequent data analysis allowed theidentification of marker polynucleotides and polypeptides whoseexpression levels were found to be highly correlated with drugresistance. Thus, in one of its embodiments, the present inventionprovides these polynucleotides and polypeptides, or “markers”, orpredictors, which show utility in predicting drug response upontreatment or exposure of cells to drug. In particular, the marker orpredictor polynucleotides and polypeptides are src biomarkerpolynucleotides and polypeptides encoding src biomarkerproteins/polypeptides.

The means of performing the gene expression and marker geneidentification analyses embraced by the present invention is describedin further detail and without limitation herein below.

IC₅₀ Determination and Phenotype Classification Based on Sensitivity ofThirty-One Colon Cell Lines to src Kinase Inhibitor Compounds

Thirty-one colon cell lines were treated with each of four src tyrosinekinase inhibitor compounds (BMS-A, BMS-B, BMS-C and BMS-D) to determinethe IC₅₀ value for each cell line. The average IC₅₀ values, along withstandard deviations, were calculated from 2 to 5 individualdeterminations for each cell line. As shown in Table 1, a largevariation in the IC₅₀ values (>1000-fold) was observed for thesecompounds among the thirty-one cell lines.

The IC₅₀ value for each cell line was log₁₀ transformed. The mean oflog₁₀(IC₅₀) across the thirty-one colon cell lines was calculated foreach compound. The value of log₁₀(C₅₀) for each cell line was comparedto the mean value of log₁₀(IC₅₀) across the thirty-one colon cell linesfor each drug. The cell lines with a log₁₀(IC₅₀) below the mean oflog₁₀(IC₅₀) were classified as sensitive to the compound, and those withan log₁₀(IC₅₀) above the mean of log₁₀(IC₅₀) were classified asresistant. Table 2 represents the resistance/sensitivity classificationsof the thirty-one colon cell lines for BMS-A, BMS-B, BMS-C and BMS-D,respectively.

As demonstrated in Table 2, the drug sensitivity classification for thethirty-one colon cell lines was the same for BMS-A and BMS-D even thoughthe IC₅₀ for these two compounds was not identical for each cell line.It was also demonstrated that most of the cell lines (26 out of 31) hadthe same resistance/sensitivity classification for all four of the srckinase inhibitor compounds tested. Five cell lines appeared to havedifferent classifications for the four src kinase inhibitor compounds asindicated in the Table 2.

Identifying Genes that Significantly Correlated with DrugResistance/Sensitivity Classification

Expression profiling data of 12,558 polynucleotides and polypeptidesrepresented on the HG-U95Av2 array for thirty-one untreated colon celllines were obtained and preprocessed as described in Example 1, Methods.The preprocessed data were analyzed using the K-mean NearestNeighborhood (KNN) algorithm to identify polynucleotides andpolypeptides whose expression patterns were strongly correlated with thedrug resistance/sensitivity classification. (Table 2). An “idealizedexpression pattern” corresponds to a gene that is uniformly high in oneclass (e.g., sensitive) and uniformly low in the other class (e.g.,resistant). Initially, a KNN analysis was performed in which acorrelation coefficient was obtained for each gene. The correlationcoefficient, which is a measure of relative classification separation,is obtained using the following formula:P(g,c)=(μ1−μ2)/(σ1+σ2).

In the above formula, for P(g,c), P represents correlation coefficient;g represents gene expression; and c represents classification.

-   -   μ1 represents the mean gene expression level of samples in class        1;    -   μ2 represents the mean gene expression level of samples in class        2;    -   σ1 represents the standard deviation of gene expression for        samples in class 1;        and σ2 represents the standard deviation of gene expression for        samples in class 2

Large values of P(g,c) indicate a strong correlation between geneexpression and resistance/sensitivity classification. When thecorrelation is compared with that in a random permutation test (randomlyassigned classification), a significance measurement is obtained. Then,the polynucleotides and polypeptides can be ranked according to thecorrelation coefficient obtained from this analysis, with the highestvalue indicating the best correlation of gene expression level with theresistance/sensitivity classification to the src kinase inhibitorcompounds in the thirty-one colon cell lines.

The KNN analysis demonstrated that many polynucleotides and polypeptidescorrelated with the drug resistance/sensitivity classification for allfour of the test compounds. Therefore, for greater stringency, twodifferent methods were applied to select a smaller subset ofpolynucleotides and polypeptides that correlated with the drugresistance/sensitive classification for all of the compounds:

First, a permutation test was performed to calculate the significance ofthe correlation coefficients obtained in the above-described KNNanalysis for the top 200 polynucleotides and polypeptides. Thosepolynucleotides and polypeptides whose ‘p’ value was less than or equalto 0.05 were selected. Second, a T-test was performed and thosepolynucleotides and polypeptides with a ‘p’ value that was equal to orless than 0.05 were selected.

Gene lists from the two analysis methods were obtained for eachcompound. When these analyses were performed, it was observed that therewere 123 polynucleotides and polypeptides as listed in Table 3 to becorrelated with the drug resistance/sensitivity classification forcompound BMS-A or BMS-D as shown in FIG. 1. Of the 123 polynucleotidesand polypeptides, 60 were highly expressed in the cell lines that wereclassified as sensitive to BMS-A or BMS-D, and 63 polynucleotides andpolypeptides were highly expressed in the cell lines that wereclassified as resistant to BMS-A or BMS-D. The same approach was used toselect polynucleotides and polypeptides (are listed in Tables 4 and 5)correlated with the drug resistance/sensitivity classification for BMS-Band BMS-C, respectively. The expression patterns of the polynucleotidesand polypeptides listed in Tables 3-5 are presented in FIGS. 1-3, whichshowed correlation with drug resistance/sensitivity classifications forthe compound BMS-A/BMS-D, BMS-B and BMS-C, respectively.

Tables 3-5 also show that 73 polynucleotides and polypeptides selectedfrom the above-described analyses are in common among all of the fourtest compounds (common polynucleotides and polypeptides are shown inTable 6). Thirty-one of the common polynucleotides and polypeptides arehighly expressed in cell lines that are classified as sensitive, and 42of the polynucleotides and polypeptides are highly expressed in celllines that are classified as resistant. Because these commonpolynucleotides and polypeptides correlate with drug sensitivity andresistance classifications, they can be used to build predictors forother biological systems as described below.

As used herein, the terms “agent” or “compounds” are meant to encompassany composition capable of modulating a protein tyrosine kinase of thepresent invention including Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn,as well as other protein tyrosine kinases, including, Bcr-abl, Jak,PDGFR, c-kit and Ephr, either directly or indirectly, and includes smallmolecule compounds, antisense reagents, antibodies, and the like.

As used herein, the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein. The term “modulate” or“modulates” is also meant to encompass an increase or decrease incellular activity, which necessarily includes a cells ability todifferentiate, proliferate, mobilize, metastasize, and/or any otheractivity that may be associated with a cells transformation into aproliferative and/or oncogenic state.

Utility of Highly Correlated Polynucleotides and Polypeptides to MakePredictions

Genes that correlate to a specific property of a biological system canbe used to make predictions about that biological system and otherbiological systems. The Genecluster software can be used to selectpolynucleotides and polypeptides and combinations of polynucleotides andpolypeptides that can predict properties using a “weighted-votingcross-validation algorithm” (T. R. Golub et al., 1999, Science,286:531-537). In particular, the Genecluster software was used to buildpredictors that demonstrate the utility of polynucleotides andpolypeptides that correlate to drug sensitivity and resistance.

As used herein, the terms “predictor” or “predictor sets” are used asfollows: a predictor refers to a single gene, or combination ofpolynucleotides and polypeptides, whose expression pattern or propertiescan be used to make predictions, with different error rates, about aproperty or characteristic of any given biological system.

The ability of gene expression patterns to predict aresistance/sensitive classification was further investigated using aWeighted Voting algorithm which uses a cross-validation strategy asdescribed by T. R. Golub et al., 1999, Science, 286:531-537. The programwas formatted to select the optimal number of polynucleotides andpolypeptides whose expression pattern could be used to predict, withoptimal accuracy, the classification of a cell line based on resistanceor sensitivity toward a src tyrosine kinase inhibitor compound, e.g.,BMS-A, BMS-B, BMS-C or BMS-D. A brief description of thecross-validation strategy of the program is described.

Based on the leave one out cross-validation strategy, a total ofthirty-one prediction analyses (i.e., the number of cell lines in thedata set) were performed in an iterative manner and the results of allthirty-one prediction analyses were combined to select the optimalnumber of polynucleotides and polypeptides that had optimal predictiveaccuracy. In each separate prediction analysis, one cell line waswithheld from the data set, and an optimal number gene predictor wasbuilt based on the remaining thirty cell lines and subsequently used topredict the class of the withheld sample.

FIG. 4 shows the real error rates using different numbers ofpolynucleotides and polypeptides in the predictor set for predictingresistant and sensitive classes to BMS-A, BMS-B, BMS-C and BMS-D in thecolon cell lines. The real error rates were compared with the real errorrates using the same number of polynucleotides and polypeptides as thepredictor set in 20 cases, in which classification for the colon celllines was randomly assigned. For example, when each predictor-setcontained 20 polynucleotides and polypeptides, the real error rate forBMS-A or BMS-D was 15.7%; for BMS-B and BMS-C, the real error rates were19% and 16.2%, respectively. This result demonstrated that these errorrate values are significantly lower than the real error rates obtainedwhen random phenotype classifications are used for the cell lines (i.e.,in a range of from 30% to 70%).

Table 7 presents a true resistance/sensitivity prediction of the 31colon cell lines for BMS-A or BMS-D, BMS-B and BMS-C using 10 markers asa predictor set (as listed in Table 10). For BMS-A or BMS-D,twenty-eight out of thirty-one cell lines were correctly predicted usingthe optimal 10-gene predictor set. Two resistant cell lines, CX-1 andSW-403, were predicted to be sensitive to BMS-A or BMS-D, while onesensitive cell lines, HCT-15, were predicted to be resistant to BMS-A orBMS-D. This resulted in a 10%% real error rate (the real error rate iscalculated by taking the average of the error rate in each class),calculated as follows:$\frac{\left( {{{2/22}\quad{resistant}} + {{1/9}\quad{sensitive}}} \right)}{2} \times 100\%\text{)}$

Different real error rates were obtained for BMS-B and for BMS-C. ForBMS-B, the optimal 10-gene predictor correctly predicted the sensitivityor resistance of 28 cell lines. The predictor made three errors. Twowrong predictions were made in the sensitive classes (calling themresistant). This resulted in an 11.6% real error rate calculated asfollows:$\frac{\left( {{{1/20}\quad{resistant}} + {{2/11}\quad{sensitive}}} \right)}{2} \times 100\%\text{)}$

For BMS-C, the optimal 10-gene predictor set predicted 29 cell linescorrectly. The predictor only made 2 errors in the sensitive classes.This resulted in an 8.3% real error rate calculated as follows:$\frac{\left( {{{0/19}\quad{resistant}} + {{2/12}\quad{sensitive}}} \right)}{2} \times 100\%\text{)}$

In addition, a confidence score for each prediction made on a cell lineby the predictor set can be obtained from the Genecluster software. Theconfidence score ranges from 0 to 1, measuring the margin of victory ineach prediction using weighted-voting algorithms (see T. R. Golub etal., 1999, Science, 286:531-537). The confidence score values for eachcell line using the optimal 10-gene predictor set obtained as describedare shown in Tables 7.

It will be appreciated that the exact number of polynucleotides andpolypeptides that should comprise an optimal predictor set is notdefinitely established or defined. It is unlikely in the real world thatany predictor set can be obtained with 100% accuracy. This is due to thefact that there is a trade-off between the amount of additionalinformation and robustness that are gained by adding morepolynucleotides and polypeptides, and the amount of noise that isconcomitantly added. In accordance with the present invention, differentnumbers of polynucleotides and polypeptides were tested in the predictorsets; data were obtained, analyzed and presented for a predictor setcomprising 10, or 15 or 25 predictor or marker polynucleotides andpolypeptides as demonstrated in Table 7-9. The selection of markerpolynucleotides and polypeptides for use in the prediction set was wellwithin the total number of polynucleotides and polypeptides thatstrongly correlated with the sensitivity class distinction (Tables 3-6).As shown in Table 8, when a predictor set comprising 15 of markerpolynucleotides and polypeptides (as listed in Table 11), the error ratefor prediction sensitivity of BMS-A/BMS-D, BMS-B and BMS-C was 12.4%, 7%and 4%, respectively. Again, different error rates were obtained when apredictor set comprising 25 of marker polynucleotides and polypeptidesas shown in Table 9 and Table 12.

Thus, in accordance with the present invention, an approach has beendeveloped in which polynucleotides and polypeptides and combinations ofpolynucleotides and polypeptides have been discovered, whose expressionpattern in a subset of cell lines correlates with, and can be used as apredictor of, in vitro response to treatment with a series of compoundsthat inhibit the function of src tyrosine kinases.

Predictor sets, error rates and algorithms used to demonstrate utility

The number of polynucleotides and polypeptides in any given predictormay influence the error rate of the predictor set in cross-validationexperiments and with other mathematical algorithms. The data show thatthe error rate of a predictor is somewhat dependent on the number ofpolynucleotides and polypeptides in the predictor set and thecontribution of each individual gene in the given predictor set and thenumber of cell lines that are tested in the cross validation experiment.For example, in a given predictor set, one gene may contribute moresignificantly than the other polynucleotides and polypeptides to theprediction.

It is very likely that if a gene significantly contributes to apredictor set, then it can be used in different combinations with otherpolynucleotides and polypeptides to achieve different error rates indifferent predictor sets, e.g., gene A alone gives an error rate of 30%.In combination with polynucleotides and polypeptides, B, C and D, theerror rate becomes 10%; in combination with polynucleotides andpolypeptides B, D and E, the error rate becomes 12%; while a combinationof gene A with polynucleotides and polypeptides E-X gives an error rateof 8%, and so on. The error rates as described herein apply to the setof cell lines used in the cross-validation experiment. If a differentset is used, or more cell lines are added to the original set tested,then different error rates may be obtained as described and understoodby the skilled practitioner. Importantly, different combinations ofpolynucleotides and polypeptides that correlate to drug sensitivity canbe used to build predictors with different prediction accuracy.

Applications of Predictor Sets

Predictor sets with different error rates may be used in differentapplications. Predictor sets can be built from any combination of thepolynucleotides and polypeptides listed in Tables 3-6, or the predictorgene subsets of 25, 15, and 7 polynucleotides and polypeptides, aspresented in Tables 7, 8, 9, 10, 11, and 12, respectively, to makepredictions about the likely effect of either src tyrosine inhibitorcompounds or compounds that affect the src tyrosine kinase signalingpathway in different biological systems. The various predictor setsdescribed herein, or the combination of these predictor sets with otherpolynucleotides and polypeptides or other co-variants of thesepolynucleotides and polypeptides, are likely to have broad utility. Forexample, they can be used as diagnostic or prognostic indicators indisease management; they can be used to predict how patients with cancermight respond to therapeutic intervention with compounds that modulatethe src tyrosine kinase family; and they can be used to predict howpatients might respond to therapeutic intervention that modulatesignaling through the entire src tyrosine kinase regulatory pathway.

While the data described herein were generated in cell lines-that areroutinely used to screen and identify compounds that have potentialutility for cancer therapy, the predictors may have both diagnostic andprognostic value in other diseases areas in which signaling throughprotein tyrosine kinases, particularly src tyrosine kinase or the srctyrosine kinase pathway is of importance, e.g., in immunology, or incancers or tumors in which cell signaling and/or proliferation controlshave gone awry. Such protein tyrosine kinases and their pathwayscomprise, for example, members of the Src family of tyrosine kinases,for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as otherprotein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit andEphr.

Further, although the data described herein have been generated usingthe particularly exemplified src tyrosine kinase inhibitor compounds,namely, BMS-A, BMS-B, BMS-C and BMS-D, the predictors may have bothdiagnostic and prognostic value related to any molecules or therapeuticinterventions that affect src tyrosine kinases or the src tyrosinekinase signaling pathways.

Those having skill in the pertinent art will appreciate that proteintyrosine kinase pathways, e.g., the Src tyrosine kinase pathway, is usedand functional in cell types other than cell lines of colon tissue.Therefore, the described predictor set of polynucleotides andpolypeptides, or combinations of polynucleotides and polypeptides withinthe predictor set, may show utility for predicting drug sensitivity orresistance to compounds that interact with or inhibit the src tyrosinekinase activity in cells from other tissues or organs associated with adisease state, or cancers or tumors derived from other tissue types.Non-limiting examples of such cells, tissues and organs include colon,breast, lung, prostate, testes, ovaries, cervix, esophagus, pancreas,spleen, liver, kidney, stomach, lymphocytic and brain, thereby providinga broad and advantageous applicability to the predictor gene setsdescribed herein. Cells for analysis can be obtained by conventionalprocedures as known in the art, for example, tissue biopsy, aspiration,sloughed cells, e.g., colonocytes, clinical or medical tissue or cellsampling procedures.

Functionality of Polynucleotides and Polypeptides that Make Up aPredictor Set

The use of a predictor, or predictor set, (e.g., predictorpolynucleotides and polypeptides, or a predictor set of polynucleotidesand polypeptides) is simply for predicting an outcome prior to havingany knowledge about a biological system. Essentially, a predictor can beconsidered to be a statistical tool. The predictor is useful primarilyin predicting the phenotype that is used to classify the biologicalsystem. In the specific embodiment provided by the present invention,the classification as “resistant” or “sensitive” is based on thelog₁₀(IC₅₀) value of each cell line to a compound (e.g., the src kinaseinhibitor compounds BMS-A, BMS-B, BMS-C or BMS-D as exemplified herein),relative to the mean log₁₀(IC₅₀) value of the cell line panel (e.g., athirty-one colon cell line panel, as exemplified herein).

A number of the polynucleotides and polypeptides as described herein(Tables 3-6) are known to be substrates for the src tyrosine kinasefamily, e.g., caveolin-1, caveolin-2, phosphoinositide 3-kinase, etc.,(M. T. Brown and J. A. Cooper, 1996, Biochemica et Biophysica Acta,1287:121-149). This is expected, since polynucleotides and polypeptidesthat contribute to a high predictor accuracy are likely to play afunctional role in the pathway that is being modulated. For example,Herceptin therapy (i.e., antibody that binds to the Her2 receptor andprevents function via internalization) is indicated when the Her2 geneis overexpressed. It is unlikely that a therapy will have anytherapeutic effect if the target enzyme is not expressed.

However, although the complete function of all of the polynucleotidesand polypeptides and their functional products (proteins and mRNAs) thatmake up a predictor set are not currently known, some of thepolynucleotides and polypeptides are likely to be directly involved inthe src tyrosine signaling pathway. In addition, some of thepolynucleotides and polypeptides in the predictor set may be indirectlyrelated to src signaling pathways. In addition, some of thepolynucleotides and polypeptides in the predictor set may function inthe metabolic or other resistance pathways specific to the compoundstested. Notwithstanding, a knowledge about the function of thepolynucleotides and polypeptides is not a requisite for determining theaccuracy of a predictor according to the practice of the presentinvention.

It has been demonstrated that different predictor sets are necessary toachieve the lowest error rate for the different compounds as testedherein. This is due to the subset of the cell lines that show differentresponses to the different compounds. Therefore, in the discoveryprocess of building a predictor, the classification of a cell as eitherresistant or sensitive to a particular compound, or series of compounds,will impact the final set of polynucleotides and polypeptides thatcomprise the best predictor/predictor set. Because differentcombinations of resistant and sensitive cells were used for eachcompound, different predictor sets were obtained. In addition, obtainingdifferent predictor sets for different compounds can be avoided if thosecell lines having common resistant or sensitive classifications of genemarker expression are use (see, e.g., the 26 cell lines presented inTable 2).

The data presented herein also reveal that there are commonpolynucleotides and polypeptides for the four different compounds (see,e.g., Table 6). It is likely that these polynucleotides and polypeptideswill have some role, whether direct or indirect, in the src tyrosinekinase pathway. Alternatively, these polynucleotides and polypeptidescan be important in intrinsically determining the sensitivity of a cellto src signaling or inhibition.

As described herein, polynucleotides and polypeptides have beendiscovered that correlate to the relative intrinsic sensitivity orresistance of colon cell lines to treatment with compounds that interactwith and inhibit src tyrosine kinases. These polynucleotides andpolypeptides have been shown, through a weighted voting cross validationprogram, to have utility in predicting the intrinsic resistance andsensitivity of colon cell lines to these compounds.

An embodiment of the present invention relates to a method ofdetermining or predicting if an individual requiring drug orchemotherapeutic treatment or therapy for a disease, for example, acancer or tumor of a particular type, will be likely to successfullyrespond or not respond to the drug or chemotherapeutic agent prior tosubjecting the individual to such treatment or chemotherapy. Preferably,the drug or chemotherapeutic agent is one that modulates proteintyrosine kinases, particularly src activity or src family tyrosinekinases activity or signaling involving src or src family tyrosinekinases. In accordance with the method of the invention, cells from atissue or organ associated with disease, e.g., a patient biopsy of atumor or cancer, preferably a colon cancer or tumor biopsy, aresubjected to an in vitro assay as described herein, to determine theirmarker gene expression pattern (polynucleotides and polypeptides fromTable 3-6) prior to their treatment with the compound or drug,particularly a protein tyrosine kinase inhibitor, preferably a srckinase inhibitor. The resulting gene expression profile of the cellsbefore drug treatment is compared with the gene expression pattern ofthe same polynucleotides and polypeptides in cells that are eitherresistant or sensitive to the drug or compound, as provided by thepresent invention, i.e., FIGS. 1-3.

Success or failure of treatment of a patient's cancer or tumor with thedrug can be determined based on the gene expression pattern of thepatient's cells being tested, compared with the gene expression patternof the predictor polynucleotides and polypeptides in the resistant orsensitive panel of that have been exposed to the drug or compound andsubjected to the predictor gene analysis detailed herein. Thus, if,following exposure to the drug, the test cells show a gene expressionpattern corresponding to that of the predictor gene set of the controlpanel of cells that are sensitive to the drug or compound, it is highlylikely or predicted that the individual's cancer or tumor will respondfavorably to treatment with the drug or compound. By contrast, if, afterdrug exposure, the test cells show a gene expression patterncorresponding to that of the predictor gene set of the control panel ofcells that are resistant to the drug or compound, it is highly likely orpredicted that the individual's cancer or tumor will not respond totreatment with the drug or compound.

As a related and more particular embodiment, the present inventionrelates to a method of determining or predicting if an individualrequiring drug or chemotherapeutic treatment or therapy for a disease,for example, a breast cancer or a breast tumor, will be likely tosuccessfully respond or not respond to the drug or chemotherapeuticagent prior to subjecting the individual to such treatment orchemotherapy. In this embodiment, the drug or chemotherapeutic agent ispreferably one that modulates src tyrosine kinase activity or signalinginvolving src tyrosine kinase. In accordance with the method of theinvention, cells from a tissue or organ associated with disease, e.g., apatient biopsy of a tumor or cancer, preferably a colon cancer or tumorbiopsy, are subjected to an in vitro assay as described herein, todetermine their marker gene expression pattern (polynucleotides andpolypeptides from Tables 3 thru 6 and/or the predictor gene subsets ofTables 10 thru 12) prior to their treatment with the src tyrosine kinaseinhibitor compound or drug. The resulting gene expression profile of thecells before drug treatment is compared with the gene expression patternof the same polynucleotides and polypeptides in cells that are eitherresistant or sensitive to the drug or compound, as provided by thepresent invention.

In another related embodiment, the present invention includes a methodof predicting, prognosing, diagnosing, and/or determining whether anindividual requiring drug therapy for a disease state orchemotherapeutic for cancer (e.g., colon cancer) will or will notrespond to treatment prior to administration of treatment. The treatmentor therapy preferably involves a protein tyrosine kinase modulatingagent, compound, or drug, for example, an inhibitor of the proteintyrosine kinase activity. Protein tyrosine kinases include, withoutlimitation, members of the Src family of tyrosine kinases, for example,Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as other proteintyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit and Ephr.Preferred is src tyrosine kinase and inhibitors thereof. In accordancewith this embodiment, cells from a patient's tissue sample, e.g., acolon tumor or cancer biopsy, are assayed to determine their geneexpression pattern prior to treatment with the protein tyrosine kinasemodulating agent, compound, or drug. The resulting gene expressionprofile of the test cells before exposure to the compound or drug iscompared with that of one or more of the predictor subsets ofpolynucleotides and polypeptides comprising either 25, 15, or 10polynucleotides and polypeptides as described herein and shown in Tables10 thru 12, respectively.

In a related embodiment, screening assays are provided for determiningif a patient's cancer or tumor is or will be susceptible or resistant totreatment with a drug or compound, particularly, a drug or compounddirectly or indirectly involved in src or src family tyrosine kinasesactivity or the src kinase pathway.

Also provided are monitoring assays to monitor the progress of a drugtreatment involving drugs or compounds that interact with or inhibitprotein tyrosine kinases, particularly src or src family tyrosinekinases activity. Protein tyrosine kinases encompassed by thesemonitoring assays include members of the Src family of tyrosine kinases,for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as otherprotein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit andEphr. Such in vitro assays are capable of monitoring the treatment of apatient having a disease treatable by a compound or agent that modulatesor interacts with a src tyrosine kinase by comparing the resistance orsensitivity gene expression pattern of cells from a patient tissuesample, e.g., a tumor or cancer biopsy, preferably a colon cancer ortumor sample, prior to treatment with a drug or compound that inhibitssrc or src family tyrosine kinases activity and again followingtreatment with the drug or compound with the expression pattern of oneor more of the predictor gene sets described, or combinations thereof.Isolated cells from the patient are assayed to determine their geneexpression pattern before and after exposure to a compound or drug,preferably a src or src family tyrosine kinases inhibitor, to determineif a change of the gene expression profile has occurred so as to warranttreatment with another drug or agent, or discontinuing currenttreatment. The resulting gene expression profile of the cells testedbefore and after treatment is compared with the gene expression patternof the predictor set of polynucleotides and polypeptides that have beendescribed and shown herein to be highly expressed in cells that areeither resistant or sensitive to the drug or compound. Alternatively, apatient's progress related to drug treatment or therapy can be monitoredby obtaining a gene expression profile as described above, only afterthe patient has undergone treatment with a given drug or therapeuticcompound. In this way, there is no need to test a patient sample priorto treatment with the drug or compound.

Such a monitoring process can indicate success or failure of a patient'streatment with a drug or compound based on the gene expression patternof the cells isolated from the patient's sample, e.g., a tumor or cancerbiopsy, as being relatively the same as or different from the geneexpression pattern of the predictor gene set of the resistant orsensitive control panel of cells that have been exposed to the drug orcompound and assessed for their gene expression profile followingexposure. Thus, if, after treatment with a drug or compound, the testcells show a change in their gene expression profile from that seenprior to treatment to one which corresponds to that of the predictorgene set of the control panel of cells that are resistant to the drug orcompound, it can serve as an indicator that the current treatment shouldbe modified, changed, or even discontinued. Also, should a patient'sresponse be one that shows sensitivity to treatment by a src or srcfamily tyrosine kinases inhibitor compound, based on correlation of theexpression profile of the predictor polynucleotides and polypeptides ofcells showing drug sensitivity with the gene expression profile fromcells from a patient undergoing treatment, the patient's treatmentprognosis can be qualified as favorable and treatment can continue.Further, if a patient has not been tested prior to drug treatment, theresults obtained after treatment can be used to determine the resistanceor sensitivity of the cells to the drug based on the gene expressionprofile compared with the predictor gene set.

In a related embodiment, the present invention embraces a method ofmonitoring the treatment of a patient having a disease treatable by acompound or agent that modulates a protein tyrosine kinase, i.e., coloncancer. Protein tyrosine kinases encompassed by such treatmentmonitoring assays include members of the Src family of tyrosine kinases,for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as otherprotein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit andEphr. For these assays, test cells from the patient are assayed todetermine their gene expression pattern before and after exposure to aprotein tyrosine kinase inhibitor compound or drug. The resulting geneexpression profile of the cells tested before and after treatment iscompared with the gene expression pattern of the predictor set ofpolynucleotides and polypeptides that have been described and shownherein to be highly expressed in cells that are either resistant orsensitive to the drug or compound. Thus, if a patient's response is orbecomes one that is sensitive to treatment by a protein tyrosine kinaseinhibitor compound, based on correlation of the expression profile ofthe predictor polynucleotides and polypeptides, the patient's treatmentprognosis can be qualified as favorable and treatment can continue.Also, if after treatment with a drug or compound, the test cells do notexhibit a change in their gene expression profile to a profile thatcorresponds to that of the control panel of cells that are sensitive tothe drug or compound, this serves as an indicator that the currenttreatment should be modified, changed, or even discontinued. Suchmonitoring processes can be repeated as necessary or desired and canindicate success or failure of a patient's treatment with a drug orcompound, based on the gene expression pattern of the cells isolatedfrom the patient's sample. The monitoring of a patient's response to agiven drug treatment can also involve testing the patient's cells in theassay as described, only after treatment, rather than before and aftertreatment, with drug or active compound.

In a preferred embodiment, the present invention embraces a method ofmonitoring the treatment of a patient having a disease treatable by acompound or agent that modulates a src tyrosine kinase, i.e., coloncancer. The test cells from the patient are assayed to determine theirgene expression pattern before and after exposure to a src tyrosinekinase inhibitor compound or drug. The resulting gene expression profileof the cells tested before and after treatment is compared with the geneexpression pattern of the predictor set of polynucleotides andpolypeptides that have been described and shown herein to be highlyexpressed in cells that are either resistant or sensitive to the drug orcompound. Thus, if a patient's response is or becomes one that issensitive to treatment by a src tyrosine kinase inhibitor compound,based on correlation of the expression profile of the predictorpolynucleotides and polypeptides, the patient's treatment prognosis canbe qualified as favorable and treatment can continue. Also, if aftertreatment with a drug or compound, the test cells do not exhibit achange in their gene expression profile to a profile that corresponds tothat of the control panel of cells that are sensitive to the drug orcompound, this serves as an indicator that the current treatment shouldbe modified, changed, or even discontinued. Such monitoring processescan be repeated as necessary or desired and can indicate success orfailure of a patient's treatment with a drug or compound, based on thegene expression pattern of the cells isolated from the patient's sample.The monitoring of a patient's response to a given drug treatment canalso involve testing the patient's cells in the assay as described onlyafter treatment, rather than before and after treatment, with drug oractive compound.

In another embodiment, the present invention encompasses a method ofclassifying whether a biological system, preferably cells from a tissue,organ, tumor or cancer of an afflicted individual, will be resistant orsensitive to a compound that modulates the system. In a preferred aspectof this invention, the sensitivity or resistance of cells, e.g., thoseobtained from a tumor or cancer, to a src tyrosine kinase inhibitorcompound, or series of compounds, is determined. According to themethod, a resistance/sensitivity profile of the cells after exposure tothe src kinase inhibitor drug or compound can be determined via geneexpression profiling protocols set forth herein. Suchresistance/sensitivity profile of the cells reflects an IC₅₀ value ofthe cells to the compound(s) as determined using a suitable assay, suchas an in vitro cytotoxicity assay as described in Example 1. A procedureof this sort can be performed using a variety of cell types andcompounds that interact with src tyrosine kinase, or affect its activityin the src or src family tyrosine kinases signaling pathway.

In another of its embodiments, the present invention contemplates thepreparation of one or more specialized microarrays (e.g.,oligonucleotide microarrays or cDNA microarrays) comprising all of thepolynucleotides and polypeptides in the Tables 3-5, or combinationsthereof, of the predictor gene sets described herein that have beendemonstrated to be most highly correlated with sensitivity (orresistance) to src or src family tyrosine kinases modulators,particularly inhibitors of src tyrosine kinase. Preferably, thepredictor gene sets are common for predicting sensitivity among morethan one src kinase modulator, e.g. a src kinase inhibitor, asdemonstrated herein. In accordance with this aspect of the invention,the oligonucleotide sequences or cDNA sequences include any of thepredictor polynucleotides and polypeptides or gene combinations asdescribed herein, which are highly expressed in resistant or sensitivecells, and are contained on a microarray, e.g., a oligonucleotidemicroarray or cDNA microarray in association with, or introduced onto,any supporting materials, such as glass slides, nylon membrane filters,glass or polymer beads, or other types of suitable substrate material.

Cellular nucleic acid, e.g., RNA, is isolated either from cellsundergoing testing after exposure to a drug or compound that interactswith src tyrosine kinase, or its signaling pathway, or from cells beingtested to obtain an initial determination or prediction of cells'sensitivity to the drug or compound, and, ultimately, a prediction oftreatment outcome with the drug or compound. The isolated nucleic acidis appropriately labeled and applied to one or more of the specializedmicroarrays. The resulting pattern of gene expression on the specializedmicroarray is analyzed as described herein and known in the art. Apattern of gene expression correlating with either sensitivity orresistance to the drug or compound is able to be determined, e.g., viacomparison with the gene expression patterns as shown in FIGS. 1-3 forthe panel of cells exposed to the src kinase inhibitors assayed herein.

In accordance with the specialized microarray embodiment of thisinvention, the microarray contains the polynucleotides and polypeptidesof one or more of the predictor gene sets, or a combination thereof, orall of the gene in the Tables 3-5, that are highly correlated with drugsensitivity or resistance by a cell type. (See, for example, Table 1 forcolon cells). If the nucleic acid target isolated from test cells, suchas tumor or cancer cells, preferably colon cancer or tumor cells, showsa high level of detectable binding to the polynucleotides andpolypeptides of the predictor set for drug sensitivity relative tocontrol, then it can be predicted that a patient's cells will respond tothe drug, or a series of drugs, and that the patient's response to thedrug, or a series of drugs, will be favorable.

Such a result predicts that the cells of a tumor or cancer are goodcandidates for the successful treatment or therapy utilizing the drug,or series of drugs. Alternatively, if the nucleic acid target isolatedfrom test cells shows a high level of detectable binding to thepolynucleotides and polypeptides of the predictor set for drugresistance, relative to control, then it can be predicted that a patientis likely not to respond to the drug, or a series of drugs, and that thepatient's response to the drug, or a series of drugs, is not likely tobe favorable. Such a result predicts that the cells of a tumor or cancerare not good candidates for treatment or therapy utilizing the drug, orseries of drugs.

The utilization of microarray technology is known practiced in the art.Briefly, to determine gene expression using microarray technology,polynucleotides, e.g., RNA, DNA, cDNA, preferably RNA, are isolated froma biological sample, e.g., cells, as described herein for colon cells.The isolated nucleic acid is detectably labeled, e.g., fluorescent,enzyme, or chemiluminescent label, and applied to a microarray, e.g.,the specialized microarrays provided by this invention. The array isthen washed to remove unbound material and visualized by staining orfluorescence, or other means known in the art depending on the type oflabel utilized.

In another embodiment of this invention, the predictor gene sets, orsubsets of polynucleotides and polypeptides comprising the predictorgene sets, can be used as biomarkers for cells that are resistant orsensitive to src kinase inhibitor compounds. With the predictorpolynucleotides and polypeptides in hand, screening and detection assayscan be carried out to determine whether or not a given compound,preferably a src kinase inhibitor compound, elicits a sensitive or aresistant phenotype following exposure of cells, e.g., a tumor or cancerbiopsy sample, such as a colon cancer cell sample, to the compound.Thus, methods of screening, monitoring, detecting, and/or diagnosing todetermine the resistance or sensitivity of cells to a drug or compoundthat interacts with src tyrosine kinase, or the src kinase pathway,preferably an inhibitor compound, and to which the cells are exposed,are encompassed by the present invention.

Such methods embrace a variety of methods and assays to determine andassess the expression of polynucleotides and polypeptides, inparticular, the predictor or src biomarker polynucleotides andpolypeptides as described herein (Tables 3-6), in cells that have beenexposed to drugs or compounds that interact with or effect a proteintyrosine kinase, or a protein tyrosine kinase pathway, wherein theprotein tyrosine kinases include members of the Src family of tyrosinekinases, for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as wellas other protein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kitand Ephr. Suitable methods include detection and evaluation of geneactivation or expression at the level of nucleic acid, e.g., DNA, RNA,mRNA, and detection and evaluation of encoded protein. For example, PCRassays as known and practiced in the art can be employed to quantify RNAin cells being assayed for susceptibility to drug treatment, forexample, src kinase inhibitors. (see Example 2, RT-PCR).

In another embodiment, the present invention is directed to a method ofidentifying cells, tissues, and/or patients that are predicted to beresistant to either protein tyrosine inhibitor compounds or compoundsthat affect protein tyrosine kinase signaling pathways, e.g., Srctyrosine kinase, or that are resistant in different biological systemsto those compounds. The method comprises the step(s) of (i) analyzingthe expression of only those polynucleotides and polypeptides listed inTables 3 thru 6, or any combination thereof, that have been shown to becorrelative to predicting resistant responses to such compounds; (ii)comparing the observed expression levels of those correlative resistantpolynucleotides and polypeptides in the test cells, tissues, and/orpatients to the expression levels of those same polynucleotides andpolypeptides in a cell line that is known to be resistant to thecompounds; and (iii) predicting whether the cells, tissues, and/orpatients are resistant to the compounds based upon the overallsimilarity of the observed expression of those polynucleotides andpolypeptides in step (ii).

In another embodiment, the present invention is directed to a method ofidentifying cells, tissues, and/or patients that are predicted to besensitive to either protein tyrosine inhibitor compounds or compoundsthat affect protein tyrosine kinase signaling pathways, e.g., the Srctyrosine kinase, or that are sensitive in different biological systemsto those compounds. The method involves the step(s) of (i) analyzing theexpression of only those polynucleotides and polypeptides listed inTables 3 thru 6, or any combination thereof, that have been shown to becorrelative to predicting sensitive responses to such compounds; (ii)comparing the observed expression levels of those correlative sensitivepolynucleotides and polypeptides in the test cells, tissues, and/orpatients to the expression levels of those same polynucleotides andpolypeptides in a cell line that is known to be sensitive to thecompounds; and (iii) predicting whether the cells, tissues, and/orpatients are sensitive to the compounds based upon the overallsimilarity of the observed expression of those polynucleotides andpolypeptides in step (ii).

The present invention further encompasses the detection and/orquantification of one or more of the protein tyrosine kinase biomarkerproteins of the present invention using antibody-based assays (e.g.,immunoassays) and/or detection systems. As mentioned herein, proteintyrosine kinase biomarkers encompass members of the Src family oftyrosine kinases, for example, Src, Fgr, Fyn, Yes, Blk Hck, Lck and Lyn,as well as other protein tyrosine kinases, including, Bcr-abl, Jak,PDGFR, c-kit and Ephr. Such assays include the following non-limitingexamples, ELISA, immunofluorescence, FACS, Western Blots, etc., asfurther described herein.

In another embodiment, the human protein tyrosine kinase biomarkerpolypeptides and/or peptides of the present invention, or immunogenicfragments or oligopeptides thereof, can be used for screeningtherapeutic drugs or compounds in a variety of drug screeningtechniques. The fragment employed in such a screening assay can be freein solution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The reduction or abolition of activity of theformation of binding complexes between the biomarker protein and theagent being tested can be measured. Thus, the present invention providesa method for screening or assessing a plurality of compounds for theirspecific binding affinity with a protein kinase biomarker polypeptide,or a bindable peptide fragment thereof, of this invention. The methodcomprises the steps of providing a plurality of compounds; combining theprotein kinase biomarker polypeptide, or a bindable peptide fragmentthereof, with each of the plurality of compounds, for a time sufficientto allow binding under suitable conditions; and detecting binding of thebiomarker polypeptide or peptide to each of the plurality of testcompounds, thereby identifying the compounds that specifically bind tothe biomarker polypeptide or peptide. More specifically, the biomarkerpolypeptide or peptide is that of a Src tyrosine kinase.

Methods of identifying compounds that modulate the activity of the humanprotein tyrosine kinase biomarker polypeptides and/or peptides areprovided by the present invention and comprise combining a potential orcandidate compound or drug modulator of protein kinase biologicalactivity with an protein kinase biomarker polypeptide or peptide, forexample, the Src tyrosine kinase biomarker amino acid sequences as setforth in Table 2, and measuring an effect of the candidate compound ordrug modulator on the biological activity of the protein kinasebiomarker polypeptide or peptide. Such measurable effects include, forexample, a physical binding interaction; the ability to cleave asuitable protein kinase substrate; effects on a native and clonedprotein kinase biomarker-expressing cell line; and effects of modulatorsor other protein kinase-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel protein tyrosine kinase biomarker polypeptides ofthe present invention comprises combining a potential or candidatecompound or drug modulator of a protein tyrosine kinase biologicalactivity, e.g., a Src tyrosine kinase, with a host cell that expressesthe protein tyrosine kinase biomarker polypeptide and measuring aneffect of the candidate compound or drug modulator on the biologicalactivity of the protein tyrosine kinase biomarker polypeptide. The hostcell can also be capable of being induced to express the proteintyrosine kinase biomarker polypeptide, e.g., via inducible expression.Physiological effects of a given modulator candidate on the proteintyrosine kinase biomarker polypeptide can also be measured. Thus,cellular assays for particular protein tyrosine kinase modulators, e.g.,a src kinase modulator, can be either direct measurement orquantification of the physical biological activity of the proteintyrosine kinase biomarker polypeptide, or they may be measurement orquantification of a physiological effect. Such methods preferably employa protein tyrosine kinase biomarker polypeptide as described herein, oran overexpressed recombinant protein tyrosine kinase biomarkerpolypeptide in suitable host cells containing an expression vector asdescribed herein, wherein the protein tyrosine kinase biomarkerpolypeptide is expressed, overexpressed, or undergoes up-regulatedexpression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa protein tyrosine kinase biomarker polypeptide, e.g., a Src kinasebiomarker polypeptide. The method comprises providing a host cellcontaining an expression vector harboring a nucleic acid sequenceencoding a protein tyrosine kinase biomarker polypeptide, or afunctional peptide or portion thereof (e.g., the src polypeptide,protein, peptide, or fragment sequences as set forth in Tables 3 thru12, or the Sequence Listing herein); determining the biological activityof the expressed protein tyrosine kinase biomarker polypeptide in theabsence of a modulator compound; contacting the cell with the modulatorcompound and determining the biological activity of the expressedprotein tyrosine kinase biomarker polypeptide in the presence of themodulator compound. In such a method, a difference between the activityof the protein tyrosine kinase biomarker polypeptide in the presence ofthe modulator compound and in the absence of the modulator compoundindicates a modulating effect of the compound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as protein tyrosine kinase modulators can be any smallchemical compound, or biological entity (e.g., protein, sugar, nucleicacid, or lipid). Test compounds are typically small chemical moleculesand peptides. Generally, the compounds used as potential modulators canbe dissolved in aqueous or organic (e.g., DMSO-based) solutions. Theassays are designed to screen large chemical libraries by automating theassay steps and providing compounds from any convenient source. Assaysare typically run in parallel, for example, in microtiter formats onmicrotiter plates in robotic assays. There are many suppliers ofchemical compounds, including, for example, Sigma (St. Louis, Mo.),Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, MO), FlukaChemika-Biochemica Analytika (Buchs, Switzerland). Also, compounds canbe synthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel protein tyrosine kinasebiomarker, e.g., src biomarker, polynucleotides and polypeptidesdescribed herein. Such high throughput screening methods typicallyinvolve providing a combinatorial chemical or peptide library containinga large number of potential therapeutic compounds (e.g., ligand ormodulator compounds). The combinatorial chemical libraries or ligandlibraries are then screened in one or more assays to identify thoselibrary members (e.g., particular chemical species or subclasses) thatdisplay a desired characteristic activity. The compounds so identifiedcan serve as conventional lead compounds, or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, prepared by combining a number of chemical building blocks(i.e., reagents such as amino acids). As an example, a linearcombinatorial library, e.g., a polypeptide or peptide library, is formedby combining a set of chemical building blocks in every possible way fora given compound length (i.e., the number of amino acids in apolypeptide or peptide compound). Millions of chemical compounds can besynthesized through such combinatorial mixing of chemical buildingblocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptoids(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad. Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagiharaet al., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J.Amer. Chem. Soc., 114:9217-9218), analogous organic synthesis of smallcompound libraries (Chen et al., 1994, J. Amer. Chem. Soc., 116:2661),oligocarbamates (Cho et al., 1993, Science, 261:1303), and/or peptidylphosphonates (Campbell et al., 1994, J. Org. Chem., 59:658), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (U.S. Pat. No. 5,539,083), antibody libraries(e.g., Vaughn et al., 1996, Nature Biotechnology, 14(3):309-314) andPCT/US96/10287), carbohydrate libraries (e.g., Liang et al., 1996,Science, 274-1520-1522) and U.S. Pat. No. 5,593,853), small organicmolecule libraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993,page 33; and U.S. Pat. No. 5,288,514; isoprenoids (U.S. Pat. No.5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No.5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134);morpholino compounds (U.S. Pat. No. 5,506,337); and the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one aspect, the invention provides solid phase-based in vitro assaysin a high throughput format, where the cell or tissue expressing atyrosine kinase protein/polypeptide/peptide is attached to a solid phasesubstrate. In such high throughput assays, it is possible to screen upto several thousand different modulators or ligands in a single day. Inparticular, each well of a microtiter plate can be used to perform aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can be used to test a single modulator. Thus, a single standardmicrotiter plate can be used in to assay about 96 modulators. If 1536well plates are used, then a single plate can easily assay from about100 to about 1500 different compounds. It is possible to assay severaldifferent plates per day; thus, for example, assay screens for up toabout 6,000-20,000 different compounds are possible using the describedintegrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a tyrosine kinase biomarker polypeptide or peptide, suchas a Src tyrosine kinase biomarker polypeptide or peptide. Particularlypreferred are assays suitable for high throughput screeningmethodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed, ingeneral, is a target protein, preferably substantially purified, and alibrary or panel of compounds (e.g., ligands, drugs, or smallmolecules), or biological entities to be screened or assayed for bindingto the protein target. Preferably, most small molecules that bind to thetarget protein modulate the target's activity in some manner due topreferential, higher affinity binding to functional areas or sites onthe protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano et al.(See also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assay allowsthe detection of small molecules (e.g., drugs, ligands) that bind toexpressed, and preferably purified, tyrosine kinase biomarkerproteins/polypeptides/peptides, such as the Src tyrosine kinase, basedon affinity of binding determinations by analyzing thermal unfoldingcurves of protein-drug or ligand complexes. The drugs or bindingmolecules determined by this technique can be further assayed, ifdesired, by methods such as those described herein to determine if themolecules affect or modulate function or activity of the target protein.

To purify a tyrosine kinase biomarker polypeptide or peptide, e.g., Srctyrosine kinase, to measure a biological binding or ligand bindingactivity, the source may be a whole cell lysate that can be prepared bysuccessive freeze-thaw cycles (e.g., one to three) in the presence ofstandard protease inhibitors. The tyrosine kinase biomarker polypeptidecan be partially or completely purified by standard protein purificationmethods, e.g., affinity chromatography using specific antibody(ies)described herein, or by ligands specific for an epitope tag engineeredinto the recombinant tyrosine kinase biomarker polypeptide molecule,also as described herein. Binding activity can then be measured asdescribed.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe tyrosine kinase biomarker polypeptides according to the presentinvention, are a preferred embodiment of this invention. It iscontemplated that such modulatory compounds can be employed in treatmentand therapeutic methods for treating a condition that is mediated by thetyrosine kinase biomarker polypeptides, e.g., Src tyrosine kinasebiomarker polypeptides, by administering to an individual in need ofsuch treatment a therapeutically effective amount of the compoundidentified by the methods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the tyrosine kinase biomarker polypeptidesof the invention, comprising administering to the individual atherapeutically effective amount of the tyrosine kinasebiomarker-modulating compound identified by a method provided herein. Inaccordance with this invention, the tyrosine kinase biomarkerpolypeptides or proteins encompassed by the methods include members ofthe Src family of tyrosine kinases, for example, Src, Fgr, Fyn, Yes,Blk, Hck, Lck and Lyn, as well as other protein tyrosine kinases,including, Bcr-abl, Jak, PDGFR, c-kit and Ephr.

The present invention particularly provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by Src biomarker polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the Src biomarker-modulating compound identified bya method provided herein.

Antibodies directed against the src biomarker proteins of the presentinvention, or antigenic or immunogenic epitopes thereof, can be, forexample, polyclonal or monoclonal antibodies. The present invention alsoincludes chimeric, single chain, and humanized antibodies, as well asFab, F(ab′)₂, or Fv fragments, or the product of an Fab expressionlibrary. Various procedures known in the art may be used for theproduction of such antibodies and antibody fragments.

Antibodies generated against the polypeptides or peptides correspondingto one or more of the src biomarker sequences of the present inventioncan be obtained by direct injection of the polypeptides or peptides intoan animal, or by administering the polypeptides or peptides to ananimal, preferably a nonhuman animal. The antibodies so obtained willthen bind to the polypeptides or peptides. In this manner, even asequence encoding only a fragment of a polypeptide can be used togenerate antibodies binding to the whole native polypeptide. Suchantibodies can be used, for example, to isolate the polypeptide fromtissue expressing that polypeptide.

For the preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunol. Today, 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole etal., 1985. In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

An ELISA assay initially involves preparing an antibody specific toantigens of the src biomarker proteins or polypeptides, preferably amonoclonal antibody. In addition, a reporter antibody is used whichrecognizes and binds to the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as a radioactive isotope,a fluorescent moiety, or, in this example, an enzyme, such ashorseradish peroxidase.

To carry out the ELISA assay, a sample is removed from a host, e.g., apatient sample, and incubated on a solid support, e.g., wells of amicrotiter plate, or a polystyrene dish, to which the proteins in thesample can bind. Any free protein binding sites on the dish are thenblocked by incubating with a non-specific protein such as bovine serumalbumin. The monoclonal antibody is then added to the solid support,e.g., the wells or the dish, and allowed to incubate. During theincubation time, the monoclonal antibodies attach to any src biomarkerproteins or polypeptides that have attached to the polystyrene dish. Allunbound monoclonal antibody is washed away using an appropriate buffersolution. The reporter antibody, e.g., linked to horseradish peroxidase,is added to the support, thereby resulting in the binding of thereporter antibody to any monoclonal antibody which has bound to srcbiomarker proteins or polypeptides that are present in the sample.Unattached reporter antibody is then washed away. Peroxidase substrateis added to the support and the amount of color developed in a giventime period provides a measurement of the amount of src biomarkerproteins or polypeptides that are present in a given volume of patientsample when compared against a standard curve.

The present invention encompasses polypeptides comprising, oralternatively, consisting of, an epitope of the polypeptide having anamino acid sequence of one or more of the src biomarker amino acidsequences as set forth in Tables 3-6. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of src biomarkers of the invention.

The term “epitopes” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope” as used herein, refers to a portion of a protein that elicitsan antibody response in an animal, as determined by any method known inthe art, for example, by the methods for generating antibodies describedinfra. (See, for example, Geysen et al., 1983, Proc. Natl, Acad. Sci.USA, 81:3998-4002). The term “antigenic epitope” as used herein refersto a portion of a protein to which an antibody can immunospecificallybind to its antigen as determined by any method well known in the art,for example, by the immunoassays described herein. Immunospecificbinding excludes non-specific binding, but does not necessarily excludecross-reactivity with other antigens. Antigenic epitopes need notnecessarily be immunogenic. Either the full-length protein or anantigenic peptide fragment can be used. Antibodies are preferablyprepared from these regions or from discrete fragments in regions of thesrc biomarker nucleic acid and protein sequences comprising an epitope.

Moreover, antibodies can also be prepared from any region of thepolypeptides and peptides of the src biomarkers as described herein. Apreferred fragment generates the production of an antibody thatdiminishes or completely prevents ligand binding. In addition,antibodies can be developed against an entire receptor or portions ofthe receptor, for example, the intracellular carboxy terminal domain,the amino terminal extracellular domain, the entire transmembranedomain, specific transmembrane segments, any of the intracellular orextracellular loops, or any portions of these regions. Antibodies canalso be developed against specific functional sites, such as the site ofligand binding, or sites that are glycosylated, phosphorylated,myristylated, or amidated, for example.

Polypeptide or peptide fragments that function as epitopes may beproduced by any conventional means. (See, e.g., Houghten, 1985, Proc.Natl. Acad. Sci. USA, 82:5131-5135; and as described in U.S. Pat. No.4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes are useful, forexample, to raise antibodies, including monoclonal antibodies, thatspecifically bind the epitope. In addition, antigenic epitopes can beused as the target molecules in immunoassays. (See, for instance, Wilsonet al., 1984, Cell, 37:767-778; and Sutcliffe et al., 1983, Science,219:660-666). Such fragments as described herein are not to beconstrued, however, as encompassing any fragments which may be disclosedprior to the invention.

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,1985, Proc. Natl. Acad. Sci. USA, 82:910-914; and Bittle et al., 1985,J. Gen. Virol., 66:2347-2354). Preferred immunogenic epitopes includethe immunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes.

Src biomarker polypeptides comprising one or more immunogenic epitopeswhich elicit an antibody response can be introduction together with acarrier protein, such as an albumin, to an animal system (such as rabbitor mouse). Alternatively, if the polypeptide is of sufficient length(e.g., at least about 25 amino acids), the polypeptide can be presentedwithout a carrier. However, immunogenic epitopes comprising as few as 5to 10 amino acids have been shown to be sufficient to raise antibodiescapable of binding to, at the very least, linear epitopes in a denaturedpolypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra; and Bittle et al., supra). If in vivo immunization is used,animals can be immunized with free peptide; however, the anti-peptideantibody titer may be boosted by coupling the peptide to amacromolecular carrier, such as keyhole limpet hemacyanin (KLH), ortetanus toxoid (IT). For instance, peptides containing cysteine residuescan be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent, such asglutaraldehyde.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs), first described by J. P. Tam et al.,1995, Biomed. Pept., Proteins, Nucleic Acids, 199, 1(3):123-32; andCalvo, et al., 1993, J. Immunol., 150(4):1403-12), which are herebyincorporated by reference in their entirety herein. MAPs containmultiple copies of a specific peptide attached to a non-immunogeniclysine core. MAP peptides usually contain four or eight copies of thepeptide, which are often referred to as MAP4 or MAP8 peptides. By way ofnon-limiting example, MAPs can be synthesized onto a lysine core matrixattached to a polyethylene glycol-polystyrene (PEG-PS) support. Thepeptide of interest is synthesized onto the lysine residues using9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For example, AppliedBiosystems (Foster City, Calif.) offers commercially available MAPresins, such as, for example, the Fmoc Resin 4 Branch and the Fmoc Resin8 Branch which can be used to synthesize MAPs. Cleavage of MAPs from theresin is performed with standard trifloroacetic acid (TFA)-basedcocktails known in the art. Purification of MAPs, except for desalting,is not generally necessary. MAP peptides can be used in immunizingvaccines which elicit antibodies that recognize both the MAP and thenative protein from which the peptide was derived.

Epitope-bearing peptides of the invention can also be incorporated intoa coat protein of a virus, which can then be used as an immunogen or avaccine with which to immunize animals, including humans, in orderstimulate the production of anti-epitope antibodies. For example, the V3loop of the gp120 glycoprotein of the human immunodeficiency virus type1 (HIV-1) has been engineered to be expressed on the surface ofrhinovirus. Immunization with rhinovirus displaying the V3 loop peptideyielded apparently effective mimics of the HIV-1 immunogens (as measuredby their ability to be neutralized by anti-HIV-1 antibodies as well asby their ability to elicit the production of antibodies capable ofneutralizing HIV-1 in cell culture). This techniques of using engineeredviral particles as immunogens is described in more detail in Smith etal., 1997, Behring Inst Mitt Feb, (98):229-39; Smith et al., 1998, J.Virol., 72:651-659; and Zhang et al., 1999, Biol. Chem., 380:365-74),which are hereby incorporated by reference herein in their entireties.

Epitope bearing polypeptides of the invention can be modified, forexample, by the addition of amino acids at the amino- and/orcarboxy-terminus of the peptide. Such modifications are performed, forexample, to alter the conformation of the epitope bearing polypeptidesuch that the epitope will have a conformation more closely related tothe structure of the epitope in the native protein. An example of amodified epitope-bearing polypeptide of the invention is a polypeptidein which one or more cysteine residues have been added to thepolypeptide to allow for the formation of a disulfide bond between twocysteines, thus resulting in a stable loop structure of theepitope-bearing polypeptide under non-reducing conditions. Disulfidebonds can form between a cysteine residue added to the polypeptide and acysteine residue of the naturally-occurring epitope, or between twocysteines which have both been added to the naturally-occurringepitope-bearing polypeptide.

In addition, it is possible to modify one or more amino acid residues ofthe naturally-occurring epitope-bearing polypeptide by substitution withcysteines to promote the formation of disulfide bonded loop structures.Cyclic thioether molecules of synthetic peptides can be routinelygenerated using techniques known in the art, e.g., as described in PCTpublication WO 97/46251, incorporated in its entirety by referenceherein. Other modifications of epitope-bearing polypeptides contemplatedby this invention include biotinylation.

For the production of antibodies in vivo, host animals, such as rabbits,rats, mice, sheep, or goats, are immunized with either free orcarrier-coupled peptides or MAP peptides, for example, byintraperitoneal and/or intradermal injection. Injection material istypically an emulsion containing about 100 μg of peptide or carrierprotein and Freund's adjuvant, or any other adjuvant known forstimulating an immune response. Several booster injections may beneeded, for instance, at intervals of about two weeks, to provide auseful titer of anti-peptide antibody which can be detected, forexample, by ELISA assay using free peptide adsorbed to a solid surface.The titer of anti-peptide antibodies in serum from an immunized animalcan be increased by selection of anti-peptide antibodies, e.g., byadsorption of the peptide onto a solid support and elution of theselected antibodies according to methods well known in the art.

As one having skill in the art will appreciate, and as discussed above,the src biomarker polypeptides of the present invention, which includethe following: e.g., members of the Src family of tyrosine kinases, suchas Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as other proteintyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit and Ephr, whichcomprise an immunogenic or antigenic epitope, can be fused to otherpolypeptide sequences. For example, the polypeptides of the presentinvention can be fused with the constant domain of immunoglobulins (IgA,IgE, IgG, IgD, or IgM), or portions thereof, e.g., CH1, CH2, CH3, or anycombination thereof, and portions thereof, or with albumin (including,but not limited to, recombinant human albumin, or fragments or variantsthereof (see, e.g., U.S. Pat. No. 5,876,969; EP Patent No. 0 413 622;and U.S. Pat. No. 5,766,883, incorporated by reference in their entiretyherein), thereby resulting in chimeric polypeptides. Such fusionproteins may facilitate purification and may increase half-life in vivo.This has been shown for chimeric proteins containing the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immununoglobulins.See, e.g., Traunecker et al., 1988, Nature, 331:84-86).

Enhanced delivery of an antigen across the epithelial barrier to theimmune system has been demonstrated for antigens (e.g., insulin)conjugated to an FcRn binding partner, such as IgG or Fc fragments (see,e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteinsthat have a disulfide-linked dimeric structure due to the IgG portiondisulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than are monomeric polypeptides, orfragments thereof, alone. See, e.g., Fountoulakis et al., 1995, J.Biochem., 270:3958-3964).

Nucleic acids encoding epitopes can also be recombined with a gene ofinterest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flagtag) to aid in detection and purification of the expressed polypeptide.For example, a system for the ready purification of non-denatured fusionproteins expressed in human cell lines has been described by Janknechtet al., (1991, Proc. Natl. Acad. Sci. USA, 88:8972-897). In this system,the gene of interest is subeloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag having six histidine residues. The tag serves as amatrix binding domain for the fusion protein. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto an Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins areselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention can be generated byemploying the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling can be employed to modulate the activities ofpolypeptides of the invention, such methods can be used to generatepolypeptides with altered activity, as well as agonists and antagonistsof the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793;5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997,Curr. Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol.,16(2):76-82; Hansson, et al., 1999, J. Mol. Biol., 287:265-76; andLorenzo and Blasco, 1998, Biotechniques, 24(2):308-313, the contents ofeach of which are hereby incorporated by reference in its entirety).

In an embodiment of the invention, alteration of polynucleotidescorresponding to one or more of the src biomarker polynucleotidesequences as set forth in Tables 3-6, and the polypeptides encoded bythese polynucleotides, can be achieved by DNA shuffling. DNA shufflinginvolves the assembly of two or more DNA segments by homologous orsite-specific recombination to generate variation in the polynucleotidesequence. In another embodiment, polynucleotides of the invention, ortheir encoded polypeptides, may be altered by being subjected to randommutapolynucleotides and polypeptidesis by error-prone PCR, randomnucleotide insertion, or other methods, prior to recombination. Inanother embodiment, one or more components, motifs, sections, parts,domains, fragments, etc., of a polynucleotide encoding a polypeptide ofthis invention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc., of one or more heterologousmolecules.

Another aspect of the present invention relates to antibodies and T-cellantigen receptors (TCRs), which immunospecifically bind to apolypeptide, polypeptide fragment, or variant one or more of the srcbiomarker amino acid sequences as set forth in Tables 3-6, and/or anepitope thereof, of the present invention (as determined by immunoassayswell known in the art for assaying specific antibody-antigen binding).

The basic antibody structural unit of an antibody or immunoglobulin isknown to comprise a tetramer. Each tetramer is composed of two identicalpairs of polypeptide chains, each pair having one “light” (about 25 kDa)and one “heavy” chain (about 50-70 kDa). The amino terminal portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids; the variable region is primarily responsible for antigenrecognition. The carboxy terminal portion of each chain defines aconstant region that is primarily responsible for immunoglobulineffector function. Immunoglobulin light chains, including human lightchains, are of the kappa and lambda types. Immunoglobulin heavy chainisotypes include IgM, IgD, IgG, IgA, and IgE. (See, generally,Fundamental Immunology, Ch. 7, Paul, W., Ed., 2nd Ed. Raven Press, N.Y.(1989), incorporated herein by reference in its entirety). The variableregions of each light/heavy chain pair form the antibody orimmunoglobulin binding site. Thus, for example, an intact IgG antibodyhas two binding sites. Except in bifunctional or bispecific antibodies,the two binding sites are the same.

The chains of an immunoglobulin molecule exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs of the heavy and the light chains of each pair arealigned by the framework regions, thus enabling binding to a specificepitope. From N-terminus to C-terminus, both the light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)); Chothia& Lesk, 1987, J. Mol. Biol., 196:901-917; or Chothia et al., 1989,Nature, 342:878-883.

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methods,including fusion of hybridomas or linking of Fab′ fragments. (See, e.g.,Songsivilai & Lachmann, 1990, Clin. Exp. Immunol., 79:315-321; Kostelnyet al., 1992, J. Immunol., 148:1547 1553). In addition, bispecificantibodies can be formed as “diabodies” (See, Holliger et al., 1993,Proc. Natl. Acad. Sci. USA, 90:6444-6448), or “Janusins” (See,Traunecker et al., 1991, EMBO J., 10:3655-3659 and Traunecker et al.,1992, Int. J. Cancer Suppl. 7:51-52-127).

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), intracellularly made antibodies (i.e., intrabodies), andepitope-binding fragments of any of the above. The term “antibody”, asused herein, refers to immunoglobulin molecules and immunologicallyactive portions or fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class or subclass(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) of immunoglobulinmolecule. In a preferred embodiment, the immunoglobulin is an IgG1isotype. In another preferred embodiment, the immunoglobulin is an IgG2isotype. In another preferred embodiment, the immunoglobulin is an IgG4isotype.

Immunoglobulins may have both a heavy and a light chain. An array ofIgG, IgE, IgM, IgD, IgA, and IgY heavy chains can be paired with a lightchain of the kappa or lambda types. Most preferably, the antibodies ofthe present invention are human antigen-binding antibodies and antibodyfragments and include, but are not limited to, Fab, Fab′ F(ab′)2, Fd,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv) and fragments comprising either a V_(L) or V_(H) domain.Antigen-binding antibody fragments, including single-chain antibodies,can comprise the variable region(s) alone or in combination with theentirety or a portion of the following: hinge region, and CH1, CH2, andCH3 domains. Also included in connection with the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, and CH1, CH2, and CH3 domains. Theantibodies of the invention may be from any animal origin includingbirds and mammals. Preferably, the antibodies are of human, murine(e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel,horse, or chicken origin. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries or fromanimals transgenic for one or more human immunoglobulin and that do notexpress endogenous immunoglobulins, as described infra and, for example,in U.S. Pat. No. 5,939,598.

The antibodies of the present invention can be monospecific, bispecific,trispecific, or of greater multispecificity. Multispecific antibodiescan be specific for different epitopes of a polypeptide of the presentinvention, or can be specific for both a polypeptide of the presentinvention, and a heterologous epitope, such as a heterologouspolypeptide or solid support material. (See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., 1991, J.Immunol., 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;5,573,920; 5,601,819; and Kostelny et al., 1992, J. Immunol.,148:1547-1553).

Antibodies of the present invention can be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) can be specified, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues, or aspresented in the sequences defined in Tables 3-6 herein. Furtherincluded in accordance with the present invention are antibodies whichbind to polypeptides encoded by polynucleotides which hybridize to apolynucleotide of the present invention under stringent, or moderatelystringent, hybridization conditions as described herein.

The antibodies of the invention (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof) canbind immunospecifically to a polypeptide or polypeptide fragment orvariant human src biomarker protein as set forth in Tables 3-6 and/ormonkey src biomarker protein.

By way of non-limiting example, an antibody can be considered to bind toa first antigen preferentially if it binds to the first antigen with adissociation constant (Kd) that is less than the antibody's Kd for thesecond antigen. In another non-limiting embodiment, an antibody can beconsidered to bind to a first antigen preferentially if it binds to thefirst antigen with an affinity that is at least one order of magnitudeless than the antibody's Ka for the second antigen. In anothernon-limiting embodiment, an antibody can be considered to bind to afirst antigen preferentially if it binds to the first antigen with anaffinity that is at least two orders of magnitude less than theantibody's Kd for the second antigen.

In another nonlimiting embodiment, an antibody may be considered to bindto a first antigen preferentially if it binds to the first antigen withan off rate (koff) that is less than the antibody's koff for the secondantigen. In another nonlimiting embodiment, an antibody can beconsidered to bind to a first antigen preferentially if it binds to thefirst antigen with an affinity that is at least one order of magnitudeless than the antibody's koff for the second antigen. In anothernonlimiting embodiment, an antibody can be considered to bind to a firstantigen preferentially if it binds to the first antigen with an affinitythat is at least two orders of magnitude less than the antibody's kofffor the second antigen.

Antibodies of the present invention can also be described or specifiedin terms of their binding affinity to a src biomarker polypeptide of thepresent invention, e.g., members of the Src family of tyrosine kinases,for example, Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, as well as otherprotein tyrosine kinases, including, Bcr-abl, Jak, PDGFR, c-kit andEphr. Preferred binding affinities include those with a dissociationconstant or Kd of less than 5×10⁻² M, 1×10⁻² M, 5×10⁻³ M, 1×10⁻³ M,5×10⁻⁴ M, or 1×10⁻⁴ M. More preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁵M, 1×10⁻⁵M, 5×10⁻⁶M,1×10⁻⁶M, 5×10⁻⁴ M, 1×10⁻⁷ M, 5×10⁻⁸ M, or 1×10⁻⁴ M. Even more preferredantibody binding affinities include those with a dissociation constantor Kd of less than 5×10⁻⁹ M, 1×10⁻⁹ M, 5×10⁻¹⁰ M, 1×10⁻¹⁰ M, 5×10⁻¹¹ M,1×10⁻¹¹ M, 5×10⁻¹² M, 1×10⁻¹² M, 5×10⁻¹³ M, 1×10⁻¹³ M, 5×10⁻¹⁴ M,1×10⁻¹⁴ M, 5×10⁻¹⁵ M, or 1×10⁻¹⁵ M.

In specific embodiments, antibodies of the invention bind to srcbiomarker polypeptides, or fragments, or variants thereof, with an offrate (koff) of less than or equal to about 5×10⁻² sec⁻¹, 1×10⁻² sec⁻¹,5×10⁻³ sec⁻¹, or 1×10⁻³ sec⁻¹. More preferably, antibodies of theinvention bind to src biomarker protein polypeptides or fragments orvariants thereof with an off rate (koff) of less than or equal to about5×10⁻⁴ sec⁻¹, 1×10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, 1×10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹,1×10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹, or 1×10⁻⁷ sec⁻¹.

In other embodiments, antibodies of the invention bind to src biomarkerpolypeptides or fragments or variants thereof with an on rate (kon) ofgreater than or equal to 1×10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 1×10⁴ M⁻¹sec⁻¹, or 5×10⁴ M⁻¹ sec⁻¹. More preferably, antibodies of the inventionbind to src biomarker polypeptides or fragments or variants thereof withan on rate greater than or equal to 1×10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹,1×10⁶ M⁻¹ sec⁻¹, 5×10⁻⁶ M sec⁻¹, or 1×10−7 M⁻¹ sec⁻¹.

The present invention also provides antibodies that competitivelyinhibit the binding of an antibody to an epitope of the invention asdetermined by any method known in the art for determining competitivebinding, for example, the immunoassays as described herein. In preferredembodiments, the antibody competitively inhibits binding to an epitopeby at least 95%, at least 90%, at least 85%, at least 80%, at least 75%,at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the src biomarker polypeptides of the present invention. For example,the present invention includes antibodies which disrupt receptor/ligandinteractions with polypeptides of the invention either partially orfully. The invention includes both receptor-specific antibodies andligand-specific antibodies. The invention also includesreceptor-specific antibodies which do not prevent ligand binding, but doprevent receptor activation. Receptor activation (i.e., signaling) canbe determined by techniques described herein or as otherwise known inthe art. For example, receptor activation can be determined by detectingthe phosphorylation (e.g., on tyrosine or serine/threonine) of thereceptor or its substrate by immunoprecipitation followed by westernblot analysis. In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in the absence of the antibody.

In another embodiment of the present invention, antibodies thatimmunospecifically bind to a src biomarker protein or a fragment orvariant thereof, comprise a polypeptide having the amino acid sequenceof any one of the heavy chains expressed by an anti-src biomarkerprotein antibody-expressing cell line of the invention, and/or any oneof the light chains expressed by an anti-src biomarker proteinantibody-expressing cell line of the invention. In another embodiment ofthe present invention, antibodies that immunospecifically bind to a srcbiomarker protein or a fragment or variant thereof, comprise apolypeptide having the amino acid sequence of any one of the V_(H)domains of a heavy chain expressed by an anti-src biomarker proteinantibody-expressing cell line, and/or any one of the V_(L) domains of alight chain expressed by an anti-src biomarker proteinantibody-expressing cell line. In preferred embodiments, antibodies ofthe present invention comprise the amino acid sequence of a V_(H) domainand V_(L) domain expressed by a single anti-src biomarker proteinantibody-expressing cell line. In alternative embodiments, antibodies ofthe present invention comprise the amino acid sequence of a V_(H) domainand a V_(L) domain expressed by two different anti-src biomarker proteinantibody-expressing cell lines.

Molecules comprising, or alternatively consisting of, antibody fragmentsor variants of the V_(H) and/or V_(L) domains expressed by an anti-srcbiomarker protein antibody-expressing cell line that immunospecificallybind to a src biomarker protein are also encompassed by the invention,as are nucleic acid molecules encoding these V_(H) and V_(L) domains,molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of a srcbiomarker protein, wherein said antibodies comprise, or alternativelyconsist of, a polypeptide having an amino acid sequence of any one, two,three, or more of the V_(H) CDRs contained in a heavy chain expressed byone or more anti-src biomarker protein antibody expressing cell lines.In particular, the invention provides antibodies that immunospecificallybind to a src biomarker protein, comprising, or alternatively consistingof, a polypeptide having the amino acid sequence of a V_(H) CDR1contained in a heavy chain expressed by one or more anti-src biomarkerprotein antibody expressing cell lines. In another embodiment,antibodies that immunospecifically bind to a src biomarker protein,comprise, or alternatively consist of, a polypeptide having the aminoacid sequence of a V_(H) CDR2 contained in a heavy chain expressed byone or more anti-src biomarker protein antibody expressing cell lines.In a preferred embodiment, antibodies that immunospecifically bind to asrc biomarker protein, comprise, or alternatively consist of, apolypeptide having the amino acid sequence of a V_(H) CDR3 contained ina heavy chain expressed by one or more anti-src biomarker proteinantibody expressing cell line of the invention. Molecules comprising, oralternatively consisting of, these antibodies, or antibody fragments orvariants thereof, that immunospecifically bind to a src biomarkerprotein or a Src biomarker protein fragment or variant thereof are alsoencompassed by the invention, as are nucleic acid molecules encodingthese antibodies, molecules, fragments and/or variants.

The present invention also provides antibodies that immunospecificiallybind to a polypeptide, or polypeptide fragment or variant of a srcbiomarker protein, wherein said antibodies comprise, or alternativelyconsist of, a polypeptide having an amino acid sequence of any one, two,three, or more of the V_(L) CDRs contained in a heavy chain expressed byone or more anti-src biomarker protein antibody expressing cell lines ofthe invention. In particular, the invention provides antibodies thatimmunospecifically bind to a src biomarker protein, comprising, oralternatively consisting of, a polypeptide having the amino acidsequence of a V_(L) CDR1 contained in a heavy chain expressed by one ormore anti-src biomarker protein antibody-expressing cell lines of theinvention. In another embodiment, antibodies that immunospecificallybind to a src biomarker protein, comprise, or alternatively consist of,a polypeptide having the amino acid sequence of a V_(L) CDR2 containedin a heavy chain expressed by one or more anti-src biomarker proteinantibody-expressing cell lines of the invention. In a preferredembodiment, antibodies that immunospecifically bind to a src biomarkerprotein, comprise, or alternatively consist of a polypeptide having theamino acid sequence of a V_(L) CDR3 contained in a heavy chain expressedby one or more anti-src biomarker protein antibody-expressing cell linesof the invention. Molecules comprising, or alternatively consisting of,these antibodies, or antibody fragments or variants thereof, thatimmunospecifically bind to a src biomarker protein or a src biomarkerprotein fragment or variant thereof are also encompassed by theinvention, as are nucleic acid molecules encoding these antibodies,molecules, fragments and/or variants.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants) that immunospecifically bind to a src biomarker proteinpolypeptide or polypeptide fragment or variant of a src biomarkerprotein, wherein the antibodies comprise, or alternatively consist of,one, two, three, or more V_(H) CDRS, and one, two, three or more V_(L)CDRS, as contained in a heavy chain or light chain expressed by one ormore anti-src biomarker protein antibody-expressing cell lines of theinvention. In particular, the invention provides antibodies thatimmunospecifically bind to a polypeptide or polypeptide fragment orvariant of a src biomarker protein, wherein the antibodies comprise, oralternatively consist of, a V_(H) CDR1 and a V_(L) CDR1, a V_(H) CDR1and a V_(L) CDR2, a V_(H) CDR1 and a V_(L) CDR3, a V_(H) CDR2 and aV_(L) CDR1, V_(H) CDR2 and V_(L) CDR2, a V_(H) CDR2 and a V_(L) CDR3, aV_(H) CDR3 and a V_(H) CDR1, a V_(H) CDR3 and a V_(L) CDR2, a V_(H) CDR3and a V_(L) CDR3, or any combination thereof, of the V_(H) CDRs andV_(L) CDRs contained in a heavy chain or light chain immunoglobulinmolecule expressed by one or more anti-src biomarker proteinantibody-expressing cell lines of the invention. In a preferredembodiment, one or more of these combinations are from a single anti-srcbiomarker protein antibody-expressing cell line of the invention.Molecules comprising, or alternatively consisting of, fragments orvariants of these antibodies that immunospecifically bind to the srcbiomarker proteins are also encompassed by the invention, as are nucleicacid molecules encoding these antibodies, molecules, fragments orvariants.

The present invention also provides nucleic acid molecules, generallyisolated, encoding an antibody of the invention (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof). In a specific embodiment, a nucleic acid molecule ofthe invention encodes an antibody (including molecules comprising, oralternatively consisting of, antibody fragments or variants thereof),comprising, or alternatively consisting of, a V_(H) domain having anamino acid sequence of any one of the V_(H) domains of a heavy chainexpressed by an anti-src biomarker protein antibody-expressing cell lineof the invention and a V_(L) domain having an amino acid sequence of alight chain expressed by an anti-src biomarker proteinantibody-expressing cell line of the invention. In another embodiment, anucleic acid molecule of the invention encodes an antibody (includingmolecules comprising, or alternatively consisting of, antibody fragmentsor variants thereof), comprising, or alternatively consisting of, aV_(H) domain having an amino acid sequence of any one of the V_(H)domains of a heavy chain expressed by an anti-src biomarker proteinantibody-expressing cell line of the invention, or a V_(L) domain havingan amino acid sequence of a light chain expressed by an anti-Srcbiomarker protein antibody-expressing cell line of the invention.

The present invention also provides antibodies that comprise, oralternatively consist of, variants (including derivatives) of theantibody molecules (e.g., the V_(H) domains and/or V_(L) domains)described herein, which antibodies immunospecifically bind to a srcbiomarker protein or fragment or variant thereof.

Standard techniques known to those of skill in the art can be used tointroduce mutations in the nucleotide sequence encoding a molecule ofthe invention, including, for example, site-directed mutapolynucleotidesand polypeptidesis and PCR-mediated mutapolynucleotides andpolypeptidesis which result in amino acid substitutions. Preferably themolecules are immunoglobulin molecules. Also, preferably, the variants(including derivatives) encode less than 50 amino acid substitutions,less than 40 amino acid substitutions, less than 30 amino acidsubstitutions, less than 25 amino acid substitutions, less than 20 aminoacid substitutions, less than 15 amino acid substitutions, less than 10amino acid substitutions, less than 5 amino acid substitutions, lessthan 4 amino acid substitutions, less than 3 amino acid substitutions,or less than 2 amino acid substitutions, relative to the reference V_(H)domain, V_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) domain, V_(L) CDR1,V_(L) CDR2, or V_(L) CDR3.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutarniic acid),uncharged polar side chains (e.g., glycine, asparagine, glutane, serine,threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan), beta-branched side chains (e.g., theonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Alternatively, mutations can be introducedrandomly along all or part of the coding sequence, such as by saturationmutapolynucleotides and polypeptidesis. The resultant mutants can bescreened for biological activity to identify mutants that retainactivity.

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or to improve hybridomaantibody production. Alternatively, non-neutral missense mutations canalter an antibody's ability to bind antigen. The location of most silentand neutral missense mutations is likely to be in the framework regions,while the location of most non-neutral missense mutations is likely tobe in the CDRs, although this is not an absolute requirement. One ofskill in the art is able to design and test mutant molecules withdesired properties, such as no alteration in antigen binding activity oralteration in binding activity (e.g., improvements in antigen bindingactivity or change in antibody specificity). Followingmutapolynucleotides and polypeptidesis, the encoded protein mayroutinely be expressed and the functional and/or biological activity ofthe encoded protein can be determined using techniques described hereinor by routinely modifying techniques known and practiced in the art.

In a specific embodiment, an antibody of the invention (including amolecule comprising, or alternatively consisting of, an antibodyfragment or variant thereof), that immunospecifically binds to srcbiomarker polypeptides or fragments or variants thereof, comprises, oralternatively consists of, an amino acid sequence encoded by anucleotide sequence that hybridizes to a nucleotide sequence that iscomplementary to that encoding one of the V_(H) or V_(L) domainsexpressed by one or more anti-src biomarker protein antibody-expressingcell lines of the invention, preferably under stringent conditions,e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C. followed by one or more washes in0.2×SSC/0.1% SDS at about 50-65° C., preferably under highly stringentconditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC atabout 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about68° C., or under other stringent hybridization conditions which areknown to those of skill in the art (see, for example, Ausubel, F. M. etal., eds., 1989, Current Protocols in Molecular Biology, Vol. L, GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York atpages 6.3.1-6.3.6 and 2.10.3). Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments orvariants thereof, with similar amino acid sequences often have similarstructure and many of the same biological activities. Thus, in oneembodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a src biomarker polypeptide orfragments or variants of a src biomarker polypeptide, comprises, oralternatively consists of, a V_(H) domain having an amino acid sequencethat is at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto the amino acid sequence of a V_(H) domain of a heavy chain expressedby an anti-src biomarker protein antibody-expressing cell line of theinvention.

In another embodiment, an antibody (including a molecule comprising, oralternatively consisting of, an antibody fragment or variant thereof),that immunospecifically binds to a src biomarker polypeptide orfragments or variants of a src biomarker protein polypeptide, comprises,or alternatively consists of, a V_(L) domain having an amino acidsequence that is at least 35%, at least 40%, at least 45%, at least 50%,at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of a V_(L) domain of a light chainexpressed by an anti-src biomarker protein antibody-expressing cell lineof the invention.

The present invention also provides antibodies (including moleculescomprising, or alternatively consisting of, antibody fragments orvariants thereof), that down-regulate the cell-surface expression of asrc biomarker protein, as determined by any method known in the art suchas, for example, FACS analysis or immunofluorescence assays. By way of anon-limiting hypothesis, such down-regulation may be the result ofantibody induced internalization of src biomarker protein. Suchantibodies can comprise, or alternatively consist of, a portion (e.g.,V_(H) CDR1, V_(H) CDR2, V_(H) CDR3, V_(L) CDR1, V_(L) CDR2, or V_(L)CDR3) of a V_(H) or V_(L) domain having an amino acid sequence of anantibody of the invention, or a fragment or variant thereof.

In another embodiment, an antibody that down-regulates the cell-surfaceexpression of a src biomarker protein comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a V_(H)domain of an antibody of the invention, or a fragment or variant thereofand a V_(L) domain of an antibody of the invention, or a fragment orvariant thereof. In another embodiment, an antibody that down-regulatesthe cell-surface expression of a src biomarker protein comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a V_(H) domain and a V_(L) domain from a single antibody (or scPv orFab fragment) of the invention, or fragments or variants thereof. Inanother embodiment, an antibody that down-regulates the cell-surfaceexpression of a src biomarker protein comprises, or alternativelyconsists of, a polypeptide having the amino acid sequence of a V_(H)domain of an antibody of the invention, or a fragment or variantthereof. In another embodiment, an antibody that down-regulates thecell-surface expression of a src biomarker protein comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a V_(L) domain of an antibody of the invention, or a fragment orvariant thereof.

In a preferred embodiment, an antibody that down-regulates thecell-surface expression of a src biomarker protein comprises, oralternatively consists of, a polypeptide having the amino acid sequenceof a V_(H) CDR3 of an antibody of the invention, or a fragment orvariant thereof. In another preferred embodiment, an antibody thatdown-regulates the cell-surface expression of a src biomarker proteincomprises, or alternatively consists of, a polypeptide having the aminoacid sequence of a V_(L) CDR3 of an antibody of the invention, or afragment or variant thereof. Nucleic acid molecules encoding theseantibodies are also encompassed by the invention.

In another preferred embodiment, an antibody that enhances the activityof a src biomarker protein, or a fragment or variant thereof, comprises,or alternatively consists of, a polypeptide having the amino acidsequence of a V_(L) CDR3 of an antibody of the invention, or a fragmentor variant thereof. Nucleic acid molecules encoding these antibodies arealso encompassed by the invention.

As nonlimiting examples, antibodies of the present invention can be usedto purify, detect, and target the polypeptides of the present invention,including both in vitro and in vivo diagnostic, detection, screening,and/or therapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe src biomarker polypeptides of the present invention in biologicalsamples. (See, e.g., Harlow et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 2nd Ed. 1988, which is incorporatedby reference herein in its entirety).

By way of another nonlimiting example, antibodies of the invention canbe administered to individuals as a form of passive immunization.Alternatively, antibodies of the present invention can be used forepitope mapping to identify the epitope(s) that are bound by theantibody. Epitopes identified in this way can, in turn, for example, beused as vaccine candidates, i.e., to immunize an individual to elicitantibodies against the naturally-occurring forms of one or more srcbiomarker proteins.

As discussed in more detail below, the antibodies of the presentinvention can be used either alone or in combination with othercompositions. The antibodies can further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus, or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention can be recombinantly fused or conjugated to moleculesthat are useful as labels in detection assays and to effector moleculessuch as heterologous polypeptides, drugs, radionuclides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995 and BP 396, 387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to theantibody. For example, without limitation, the antibody derivativesinclude antibodies that have been modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including, but notlimited to, specific chemical cleavage, acetylation, formylation,metabolic synthesis of tunicamycin, etc. Additionally, the derivativecan contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies directed against anantigen or immunogen of interest can be produced by various procedureswell known in the art. For example, a src biomarker polypeptide orpeptide of the invention can be administered to various host animals aselucidated above to induce the production of sera containing polyclonalantibodies specific for the antigen. Various adjuvants may be used toincrease the immunological response, depending on the host species;adjuvants include, but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum. Such adjuvants are also well known in theart.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art, including the use of hybridoma, recombinant and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques as known andpracticed in the art and as taught, for example, in Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd Ed. 1988; Hammerling, et al., In: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pages 563-681, 1981, the contents of whichare incorporated herein by reference in their entireties. The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. The term “monoclonal antibody”refers to an antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anonlimiting example, mice can be immunized with a polypeptide or peptideof the invention, or with a cell expressing the polypeptide or peptide.Once an immune response is detected, e.g., antibodies specific for theantigen are detected in the sera of immunized mice, the spleen isharvested and splenocytes are isolated. The splenocytes are then fusedby well known techniques to any suitable myeloma cells, for examplecells from cell line SP2/0 or P3×63-AG8.653 available from the ATCC.Hybridomas are selected and cloned by limiting dilution techniques. Thehybridoma clones are then assayed by methods known in the art todetermine and select those cells that secrete antibodies capable ofbinding to a polypeptide of the invention. Ascites fluid, whichgenerally contains high levels of antibodies, can be generated byimmunizing mice with positive hybridoma clones.

Accordingly, the present invention encompasses methods of generatingmonoclonal antibodies, as well as the antibodies produced by thesemethods, comprising culturing a hybridoma cell secreting an antibody ofthe invention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with a src biomarkerpolypeptide or peptide antigen of the invention with myeloma cells andthen screening the hybridomas resulting from the fusion for hybridomaclones that secrete an antibody that binds to a polypeptide of theinvention. Another well known method for producing both polyclonal andmonoclonal human B cell lines is transformation using Epstein Barr Virus(EBV). Protocols for generating EBV-transformed B cell lines arecommonly known in the art, such as, for example, the protocol outlinedin Chapter 7.22 of Current Protocols in Immunology, Coligan et al.,Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated byreference herein in its entirety. The source of B cells fortransformation is commonly human peripheral blood, but B cells fortransformation can also be obtained from other sources including, butnot limited to, lymph node, tonsil, spleen, tumor tissue, and infectedtissues. Tissues are generally prepared as single cell suspensions priorto EBV transformation. In addition, T cells that may be present in the Bcell samples can be either physically removed or inactivated (e.g., bytreatment with cyclosporin A). The removal of T cells is oftenadvantageous, because T cells from individuals seropositive for anti-EBVantibodies can suppress B cell immortalization by EBV. In general, asample containing human B cells is innoculated with EBV and cultured for3-4 weeks. A typical source of EBV is the culture supernatant of theB95-8 cell line (ATCC; VR-1492). Physical signs of EBV transformationcan generally be seen toward the end of the 3-4 week culture period.

By phase-contrast microscopy, transformed cells appear large, clear and“hairy”; they tend to aggregate in fight clusters of cells. Initially,EBV lines are generally polyclonal. However, over prolonged periods ofcell culture, EBV lines can become monoclonal as a result of theselective outgrowth of particular B cell clones. Alternatively,polyclonal EBV transformed lines can be subcloned (e.g., by limitingdilution) or fused with a suitable fusion partner and plated at limitingdilution to obtain monoclonal B cell lines. Suitable fusion partners forEBV transformed cell lines include mouse myeloma cell lines (e.g.,SP2/0, X63-Ag8.653), heteromyeloma cell lines (human×mouse; e.g.,SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500,SKO-007, RPMI 8226, and KR-4). Thus, the present invention also includesa method of generating polyclonal or monoclonal human antibodies againstpolypeptides of the invention or fragments thereof, comprisingEBV-transformation of human B cells.

Antibody fragments that recognize specific epitopes can be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F (ab′) 2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

Antibodies encompassed by the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular embodiment, such phage can be utilized to displayantigen binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds to the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured onto a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present. invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J.Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur. J.Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton etal., 1994, Advances in Immunology, 57:191-280; PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108, each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., 1992, BioTechniques,12(6):864-869; Sawai et al., 1995, AJRI, 34:2634; and Better et al.,1988, Science, 240:1041-1043, which are hereby incorporated by referenceherein in their entireties.

Examples of techniques that can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology, 203:46-88; Shu etal., 1993, Proc. Natl. Acad. Sci. USA, 90:7995-7999; and Skerra et al.,1988, Science, 240:1038-1040. For some uses, including the in vivo useof antibodies in humans and in in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. (See, e.g., Morrison, 1985, Science,229:1202; Oi et al., 1986, BioTechniques, 4:214; Gillies et al., 1989,J. Immunol. Methods, 125:191-202; and U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entirety).

Humanized antibodies are antibody molecules from non-human speciesantibody that bind to the desired antigen and have one or morecomplementarity determining regions (CDRs) from the nonhuman species andframework regions from a human immunoglobulin molecule. Often, frameworkresidues in the human framework regions are substituted with thecorresponding residues from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding, and by sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties). Antibodies can be humanized using a variety of techniquesknown in the art, including, for example, CDR-grafting (EP 239,400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089); veneering or resurfacing (EP 592,106; EP 519,596; Padlan,1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994,Protein Engineering, 7(6):805-814; Roguska et al., 1994, Proc. Natl.Acad. Sci. USA, 91:969-973; and chain shuffling (U.S. Pat. No.5,565,332).

Completely human antibodies can be made by a variety of methods known inthe art, including the phage display methods described above, usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients, so as to avoid or alleviate immune reactionto foreign protein. Human antibodies can be made by a variety of methodsknown in the art, including the phage display methods described above,using antibody libraries derived from human immunoglobulin sequences.See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publicationsWO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin polynucleotides and polypeptides. Forexample, the human heavy and light chain immunoglobulin gene complexescan be introduced randomly, or by homologous recombination, into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells, in addition to the human heavy and light chain polynucleotidesand polypeptides. The mouse heavy and light chain immunoglobulinpolynucleotides and polypeptides can be rendered nonfunctionalseparately or simultaneously with the introduction of humanimmunoglobulin loci by homologous recombination. In particular,homozygous deletion of the J_(H) region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of a polypeptide of theinvention.

Monoclonal antibodies directed against the antigen can be obtained fromthe immunized transgenic mice using conventional hybridoma technology.The human immunoglobulin transpolynucleotides and polypeptides harboredby the transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation.

Thus, using such a technique, it is possible to produce useful humanIgG, IgA, IgM and IgE antibodies. For an overview of the technology forproducing human antibodies, see Lonberg and Huszar, 1995, Intl. Rev.Immunol., 13:65-93. For a detailed discussion of the technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies, see, e.g., PCT publications WO 98/24893;WO 92101047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877;U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and6,114,598, which are incorporated by reference herein in their entirety.In addition, companies such as Abgenix, Inc. (Fremont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to theabove described technologies.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection”. In thisapproach, a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., 1988, BioTechnology,12:899-903).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotypic antibodies that “mimic” srcbiomarker polypeptides of the invention using techniques well known tothose skilled in the art. (See, e.g., Greenspan and Bona, 1989, FASEBJ., 7(5):437-444 and Nissinoff, 1991, J. Immunol., 147(8):2429-2438).For example, antibodies which bind to and competitively inhibitpolypeptide multimerization and/or binding of a polypeptide of theinvention to a ligand can be used to generate anti-idiotypes that“mimic” the polypeptide multimerization and/or binding domain and, as aconsequence, bind to and neutralize the polypeptide and/or its ligand,e.g., in therapeutic regimens. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used to neutralize polypeptideligand. For example, such anti-idiotypic antibodies can be used to binda polypeptide of the invention and/or to bind its ligands/receptors, andthereby activate or block its biological activity.

Intrabodies are antibodies, often scFvs, that are expressed from arecombinant nucleic acid molecule and are engineered to be retainedintracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum,or periplasm of the host cells). Intrabodies can be used, for example,to ablate the function of a protein to which the intrabody binds. Theexpression of intrabodies can also be regulated through the use ofinducible promoters in the nucleic acid expression vector comprisingnucleic acid encoding the intrabody. Intrabodies of the invention can beproduced using methods known in the art, such as those disclosed andreviewed in Chen et al., 1994, Hum. Gene Ther., 5:595-601; Marasco, W.A., 1997, Gene Ther., 4:11-15; Rondon and Marasco, 1997, Annu. Rev.Microbiol., 51:257-283; Proba et al., 1998, J. Mol. Biol., 275:245-253;Cohen et al., 1998, Oncogene, 17:2445-2456; Ohage and Steipe, 1999, J.Mol. Biol., 291:1119-1128; Ohage et al., 1999,J. Mol. Biol.,291:1129-1134; Wirtz and Steipe, 1999, Protein Sci., 8:2245-2250; Zhu etal., 1999, J. Immunol. Methods, 231:207-222.

XenoMouse Technology Antibodies in accordance with the invention arepreferably prepared by the utilization of a transgenic mouse that has asubstantial portion of the human antibody producing genome inserted, butthat is rendered deficient in the production of endogenous murineantibodies (e.g., XenoMouse strains available from Abgenix Inc.,Fremont, Calif.). Such mice are capable of producing humanimmunoglobulin molecules and antibodies and are virtually deficient inthe production of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving the same are disclosed in thepatents, applications, and references disclosed herein.

The ability to clone and reconstruct megabase-sized human loci in YACsand to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci, as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression. An importantpractical application of such a strategy is the “humanization” of themouse humoral immune system. Introduction of human immunoglobulin (Ig)loci into mice in which the endogenous Ig polynucleotides andpolypeptides have been inactivated offers the opportunity to study themechanisms underlying programmed expression and assembly of antibodiesas well as their role in B cell development. Furthermore, such astrategy could provide an ideal source for the production of fully humanmonoclonal antibodies (Mabs) an important milestone toward fulfillingthe promise of antibody therapy in human disease.

Fully human antibodies are expected to minimize the immunogenic andallergic responses intrinsic to mouse or mouse-derivatized monoclonalantibodies and thus to increase the efficacy and safety of theadministered antibodies. The use of fully human antibodies can beexpected to provide a substantial advantage in the treatment of chronicand recurring human diseases, such as cancer, which require repeatedantibody administrations.

One approach toward this goal was to engineer mouse strains deficient inmouse antibody production to harbor large fragments of the human Ig lociin anticipation that such mice would produce a large repertoire of humanantibodies in the absence of mouse antibodies. Large human Ig fragmentswould preserve the large variable gene diversity as well as the properregulation of antibody production and expression. By exploiting themouse machinery for antibody diversification and selection and the lackof immunological tolerance to human proteins, the reproduced humanantibody repertoire in these mouse strains should yield high affinityantibodies against any antigen of interest, including human antigens.Using the hybridoma technology, antigen-specific human monoclonalantibodies with the desired specificity could be readily produced andselected.

This general strategy was demonstrated in connection with the generationof the first “XenoMouseT” strains as published in 1994. See Green etal., 1994, Nature Genetics, 7:13-21. The XenoMouse strains wereengineered with yeast artificial chromosomes (YACS) containing 245 kband 10 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus, respectively, which containedcore variable and constant region sequences. Id. The human Ig containingYACs proved to be compatible with the mouse system for bothrearrangement and expression of antibodies and were capable ofsubstituting for the inactivated mouse Ig polynucleotides andpolypeptides. This was demonstrated by their ability to induce B-celldevelopment, to produce an adult-like human repertoire of fully humanantibodies, and to generate antigen-specific human monoclonalantibodies. These results also suggested that introduction of largerportions of the human Ig loci containing greater numbers of Vpolynucleotides and polypeptides, additional regulatory elements, andhuman Ig constant regions might recapitulate substantially the fullrepertoire that is characteristic of the human humoral response toinfection and immunization. The work of Green et al. was recentlyextended to the introduction of greater than approximately 80% of thehuman antibody repertoire through the use of megabase-sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively, to produce XenoMouse mice. See Mendez etal., 1997, Nature Genetics, 15:146-156; Green and Jakobovits, 1998, J.Exp. Med., 188:483495; and Green, 1999, Journal of ImmunologicalMethods, 231:11-23, the disclosures of which are hereby incorporatedherein by reference.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies typically are comprised of a human constant region and amurine variable region, it is expected that certain human anti-chimericantibody (HACA) responses will be observed, particularly in treatmentsinvolving chronic or multi-dose utilizations of the antibody. Thus, itis desirable to provide fully human antibodies against src biomarkerpolypeptides in order to vitiate concerns and/or effects of HAMA or HACAresponses.

Polypeptide antibodies of the invention may be chemically synthesized orproduced through the use of recombinant expression systems. Accordingly,the invention further embraces polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, anantibody that specifically binds to a polypeptide of the invention,preferably, an antibody that binds to a polypeptide having the aminoacid sequence of one or more of the src biomarker sequences as set forthin Tables 3-6.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques, 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, the annealing and ligating of thoseoligonucleotides, and then the amplification of the ligatedoligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody can be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin can be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, (or a nucleic acid, preferably poly A+ RNA, isolated from), anytissue or cells expressing the antibody, such as hybridoma cellsselected to express an antibody of the invention by PCR amplificationusing synthetic primers hybridizable to the 3′ and 5′ ends of thesequence. Alternatively, cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody can be employed. Amplifiednucleic acids generated by PCR can then be cloned into replicablecloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody are determined, the nucleotide sequence of the antibody canbe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutapolynucleotides and polypeptidesis, PCR, etc. (see, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; and Ausubel et al., eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY, which are both incorporated byreference herein in their entireties), to generate antibodies having adifferent amino acid sequence, for example, to create amino acidsubstitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains can be inspected to identify the sequencesof the CDRs by methods that are well known in the art, e.g., bycomparison to known amino acid sequences of other heavy and light chainvariable regions, to determine the regions of sequence hypervariability.Using routine recombinant DNA techniques, one or more of the CDRs can beinserted within framework regions, e.g., into human framework regions,to humanize a non-human antibody, as described supra. The frameworkregions can be naturally occurring or consensus framework regions, andpreferably, are human framework regions (see, e.g., Chothia et al.,1998, J. Mol. Biol.; 278:457-479 for a listing of human frameworkregions).

Preferably, the polynucleotide generated by the combination of theframework regions and CDRs encodes an antibody that specifically bindsto a src biomarker polypeptide of the invention. Also preferably, asdiscussed supra, one or more amino acid substitutions can be made withinthe framework regions; such amino acid substitutions are performed withthe goal of improving binding of the antibody to its antigen. Inaddition, such methods can be used to make amino acid substitutions ordeletions of one or more variable region cysteine residues participatingin an intrachain disulfide bond to generate antibody molecules lackingone or more intrachain disulfide bonds. Other alterations to thepolynucleotide are encompassed by the present invention and are withinthe skill of the art.

For some uses, such as for in vitro affinity maturation of an antibodyof the invention, it is useful to express the V_(H) and V_(L) domains ofthe heavy and light chains of one or more antibodies of the invention assingle chain antibodies, or Fab fragments, in a phage display libraryusing phage display methods as described supra. For example, the cDNAsencoding the V_(H) and V_(L) domains of one or more antibodies of theinvention can be expressed in all possible combinations using a phagedisplay library, thereby allowing for the selection of V_(H)/V_(L)combinations that bind to the src biomarker polypeptides according tothe present invention with preferred binding characteristics such asimproved affinity or improved off rates. In addition, V_(H) and V_(L)segments, particularly, the CDR regions of the V_(H) and V_(L) domainsof one or more antibodies of the invention, can be mutated in vitro.Expression of V_(H) and V_(L) domains with “mutant” CDRs in a phagedisplay library allows for the selection of V_(H)/V_(L) combinationsthat bind to src biomarker proteins which are receptor polypeptides withpreferred binding characteristics such as improved affinity or improvedoff rates.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding the V_(H) and V_(L)domains are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or from synthetic cDNA libraries.The DNA encoding the V_(H) and V_(L) domains are joined together by anscFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6or pComb 3 HSS). The vector is introduced into E. coli viaelectroporation and the E. coli is infected with helper phage. Phageused in these methods are typically filamentous phage, including fd andM13, and the V_(H) and V_(L) domains are usually recombinantly fusedeither to the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to an antigen of interest (i.e., a srcbiomarker polypeptide or a fragment thereof) can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured onto a solid surface or bead.

The antibodies according to the invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis, by intracellular immunization (i.e., intrabodytechnology), or preferably, by recombinant expression techniques.Methods of producing antibodies include, but are not limited to,hybridoma technology, EBV transformation, and other methods discussedherein as well as through the use recombinant DNA technology, asdiscussed below.

Recombinant expression of an antibody of the invention, or fragment,derivative, variant or analog thereof, (e.g., a heavy or light chain ofan antibody of the invention or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Methods for preparinga protein by expressing a polynucleotide encoding an antibody aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus embraces replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors can includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody can be cloned into such a vector for expression of the entireheavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host expression vector systems can be utilized to expressthe antibody molecules of the invention. Such expression systemsrepresent vehicles by which the coding sequences of interest can beexpressed, their encoded products produced and subsequently purified.These systems also represent cells which can, when transformed ortransfected with the appropriate nucleotide coding sequences, express anantibody molecule of the invention in situ. Cell expression systemsinclude, but are not limited, to microorganisms such as bacteria (e.g.,E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces or Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus(TMV)), transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as E. coli, and morepreferably, eukaryotic cells, especially for the expression of wholerecombinant antibody molecules, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells, in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus, is an effective expression system for antibodies(Foecking et al., 1986, Gene, 45:101; Cockett et al., 1990,BioTechnology, 8:2).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of an antibody molecule, for example, vectors that directthe expression of high levels of fusion protein products that arereadily purified are often desirable. Such vectors include, but are notlimited to, the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J., 2:1791), in which the antibody coding sequence can be ligatedindividually into the vector in-frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res., 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem., 24:5503-5509; and the like). pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign polynucleotides andpolypeptides. The virus grows in Spodoptera figuriperda cells. Theantibody coding sequence may be cloned individually into non-essentialregions (for example the polyhedrin gene) of the virus and placed undercontrol of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral based expression systems canbe utilized. In cases in which an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (See, e.g., Loganand Shenk, 1984, Proc. Natl. Acad. Sci. USA, 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in-phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol., 153:51-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product can be used. Suchmammalian host cells include, but are not limited to, CHO, VERY, BHK,Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer celllines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, andnormal mammary gland cell lines such as, for example, CRL7030 andHs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule can be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoters, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, such geneticallyengineered cells can be allowed to grow for 1-2 days in an enrichedmedium, and then are typically replated in a selective medium. Aselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which, in turn, can be cloned andexpanded into cell lines. This method can advantageously be used toengineer cell lines expressing the antibody molecule. Such engineeredcell lines are particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

A number of selection systems can be used, including but not limited to,herpes simplex virus thymidine kinase (HSV TK), (Wigler et al., 1977,Cell, 11:223), hypoxanthine-guanine phosphoribosyltransferase (HGPRT),(Szybalska and Szybalski, 1992, Proc. Natl. Acad. Sci. USA, 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell, 22:817)polynucleotides and polypeptides can be employed in tk-, hgprt-, oraprt-cells (APRT), respectively.

In addition, anti-metabolite resistance can be used as the basis ofselection for the following polynucleotides and polypeptides: dhfr,which confers resistance to methotrexate (Wigler et al., 1980, Proc.Natl. Acad. Sci. USA, 77:357; and O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA, 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA, 18:2072); neo,which confers resistance to the aminoglycoside G418 (Clinical Pharmacy,12:488-505; Wu and Wu, 1991, Biotherapy, 3:87-95; Tolstoshev, 1993, Ann.Rev. Pharmacol. Toxicol., 32:573-596; Mulligan, 1993, Science,260:926-932; Anderson, 1993, Ann. Rev. Biochem, 62:191-21; May, 1993,TIB TECH, 11(5):155-215; and hygro, which confers resistance tohygromycin (Santerre et al., 1984, Gene, 30:147). Methods commonly knownin the art of recombinant DNA technology can be routinely applied toselect the desired recombinant clone, and such methods are described,for example, in Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1993); Kriegler, 1990, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY; in Chapters 12 and13, Dracopoli et al. (eds), Current Protocols in Human Genetics, JohnWiley & Sons, NY (1994); Colberre-Garapin et al., 1981. J. Mol. Biol.,150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentscbel, The use ofvectors based on gene amplification for the expression of clonedpolynucleotides and polypeptides in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987). When a marker in the vector systemexpressing an antibody is amplifiable, an increase in the level ofinhibitor present in the host cell culture will increase the number ofcopies of the marker gene. Since the amplified region is associated withthe antibody gene, production of the antibody will also increase (Crouseet al., 1983, Mol. Cell. Biol., 3:257).

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoxirine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availability of cell lines (e.g., themurine myeloma cell line, NSO) which are glutamine synthase negative.Glutamine synthase expression systems can also function in glutaminesynthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) byproviding additional inhibitor to prevent the functioning of theendogenous gene.

Vectors that express glutamine synthase as the selectable markerinclude, but are not limited to, the pEE6 expression vector described inStephens and Cockett, 1989, Nucl. Acids. Res., 17:7110. A glutaminesynthase expression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are incorporated by reference herein in theirentireties. In addition, glutamine synthase expression vectors that canbe used in accordance with the present invention are commerciallyavailable from suppliers, including, for example, Lonza Biologics, Inc.(Portsmouth, N.H.). The expression and production of monoclonalantibodies using a GS expression system in murine myeloma cells isdescribed in Bebbington et al., 1992, BioTechnology, 10:169 and inBiblia and Robinson, 1995, Biotechnol. Prog., 11:1, which areincorporated by reference herein in their entireties.

A host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors can contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector can be used which encodes, and is capable of expressing,both the heavy and light chain polypeptides. In such situations, thelight chain should be placed before the heavy chain to avoid an excessof toxic free heavy chain (Proudfoot, 1986, Nature, 322:52; Kohler,1980, Proc. Natl. Acad. Sci. USA, 77:2197). The coding sequences for theheavy and light chains can comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it can bepurified by any method known in the art for the purification of animmunoglobulin or polypeptide molecule, for example, by chromatography(e.g., ion exchange, affinity, particularly by affinity for the specificantigen, Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies that are recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugated) to a polypeptide (or portion thereof,preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 aminoacids of the polypeptide) of the present invention to generate fusionproteins. The fusion does not necessarily need to be direct, but canoccur through linker sequences. The antibodies can be specific forantigens other than polypeptides (or portions thereof, preferably atleast 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention. For example, antibodies can beused to target the polypeptides of the present invention to particularcell types, either in vitro or in vivo, by fusing or conjugating thepolypeptides of the present invention to antibodies specific forparticular cell surface receptors.

Polypeptides and/or antibodies of the present invention (includingfragments or variants thereof) can be fused to either the N-terminal orC-terminal end of the heterologous protein (e.g., immunoglobulin Fcpolypeptide or human serum albumin polypeptide). Antibodies of theinvention can also be fused to albumin (including, but not limited to,recombinant human serum albumin (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999; EP Patent 0 413 622; and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, incorporated herein by reference in theirentirety), resulting in chimeric polypeptides. In a preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094, which is hereinincorporated by reference in its entirety). In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883 incorporated herein byreference in its entirety.

Polynucleotides encoding src biomarker fusion proteins and antibodiesthereto are also encompassed by the invention. Such fusion proteins may,for example, facilitate purification and may increase half-life in vivo.Antibodies fused or conjugated to the polypeptides of the presentinvention may also be used in in vitro immunoassays and purificationmethods using methods known in the art. See, e.g., Harbor et al., supra,and PCT publication WO 93/21232; EP 439, 095; Naramura et al., 1994,Immunol. Lett., 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al., 1992,Proc. Natl. Acad. Sci USA, 89:1428-1432; Fell et al., 1991, J. Immunol.,146:2446-2452, which are incorporated by reference herein in theirentireties.

The present invention further includes compositions comprising the srcbiomarker polypeptides of the present invention fused or conjugated toantibody domains other than the variable region domain. For example, thepolypeptides of the present. invention can be fused or conjugated to anantibody Fc region, or portion thereof. The antibody portion fused to apolypeptide of the present invention can comprise the constant region,hinge region, CH1 domain, CH2 domain, CH3 domain, or any combination ofwhole domains or portions thereof. The polypeptides can also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. (See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., 1991, Proc.Natl. Acad. Sci. USA, 88:10535-10539; Zheng et al., 1995, J. Immunol.,154:5590-5600; and Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, which are hereby incorporated by reference herein intheir entireties).

As discussed supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of one or more of the src biomarkeramino acid sequences as set forth in Tables 3-6 can be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides, or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to one or more ofthe src biomarker sequences as set forth in Tables 3-6 can be fused orconjugated to the above antibody portions to facilitate purification.For guidance, chimeric proteins having the first two domains of thehuman CD4 polypeptide and various domains of the constant regions of theheavy or light chains of mammalian immunoglobulins have been described.(EP 394,827; Traunecker et al., 1988, Nature, 331:84-86). Thepolypeptides of the present invention fused or conjugated to anantibody, or portion thereof, having disulfide-linked dimeric structures(due to the IgG), for example, can also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., 1995, J. Biochem.,270:3958-3964). In many cases, the Fc portion in a fusion protein isbeneficial in therapy, diagnosis, and/or screening methods, and thus canresult in, for example, improved pharmacokinetic properties. (EP A 232,262). In drug discovery, for example, human proteins, such as hIL-5,have been fused with Fc portions for the purpose of high-throughputscreening assays to identify antagonists of hIL-5. (See, Bennett et al.,1995, J. Molecular Recognition, 8:52-58; and Johanson et al., 1995, J.Biol. Chem., 270:9459-9471). Alternatively, deleting the Fc portionafter the fusion protein has been expressed, detected, and purified, maybe desired. For example, the Fc portion may hinder therapy and diagnosisif the fusion protein is used as an antigen for immunizations.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide, to facilitate theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., Chatsworth, Calif.), among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA, 86:821-824, for instance, hexa histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutinin (HA)protein (Wilson et al., 1984, Cell, 37:767) and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure, forexample, to determine the efficacy of a given treatment regimen.Detection can be facilitated by coupling the antibody to a detectablesubstance. Nonlimiting examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance can becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. (See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the presentinvention).

Nonlimiting examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;Nonlimiting examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; nonlimiting examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; a nonlimiting example of a luminescentmaterial includes luminol; nonlimiting examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and nonlimitingexamples of suitable radioactive material include iodine (¹²⁵I, ¹³¹I,carbon (¹⁴C), sulfur (3sus), tritium (³H), indium (¹¹¹In and otherradioactive isotopes of inidium), technetium (⁹⁹Tc, ^(99m)Tc), thallium(20′Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁹F), ¹⁵³Sm, ¹⁷⁷Lu, Gd, radioactive Pm,radioactive La, radioactive Yb, ¹⁶⁶Ho, ⁹⁰Y, radioactive Sc, radioactiveRe, radioactive Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

In specific embodiments, the src biomarker polypeptides of the inventionare attached to macrocyclic chelators useful for conjugating radiometalions, including, but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ionassociated with the macrocyclic chelators attached to the src biomarkerpolypeptides of the invention is ¹¹¹In. In another preferred embodiment,the radiometal ion associated with the macrocyclic chelator attached tothe src biomarker polypeptides. of the invention is ⁹⁰Y. In specificembodiments, the macrocyclic chelator is 1, 4, 7,10-tetraazacyclododecane-N, N′, N″, N′″-tetraacetic acid (DOTA). Inother specific embodiments, the DOTA is attached to the src biomarkerpolypeptides of the invention via a linker molecule.

Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art. (See, for example, DeNardo etal., 1998, Clin. Cancer Res., 4(10):2483-90; Peterson et al., 1999,Bioconjug. Chem., 10(4):553-557; and Zimmerman et al, 1999, Nucl. Med.Biol., 26(8):943-950, which are hereby incorporated by reference intheir entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065,which disclose chelating agents that can be conjugated to antibodies andmethods for making and using them, are hereby incorporated by referencein their entireties. Though U.S. Pat. Nos. 5,652,361 and 5,756,065 focuson conjugating chelating agents to antibodies, one skilled in the artcan readily adapt the methods disclosed therein in order to conjugatechelating agents to other polypeptides.

Antibodies can also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, In: Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56, Alan R. Liss,Inc., 1985; Hellstrom et al., “Antibodies For Drug Delivery”, In:Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53,Marcel Deldcer, Inc., 1987; Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, In: Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506, 1985; “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, In:Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-316, Academic Press, 1985; and Thorpe et al., 1982, “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-158. Alternatively, an antibody can be conjugatedto a second antibody to form an antibody heteroconjugate, e.g., asdescribed in U.S. Pat. No. 4,676,980 to Segal, which is incorporatedherein by reference in its entirety. An antibody, i.e., an antibodyspecific for a src biomarker polypeptide of this invention, with orwithout a therapeutic moiety conjugated to it, and administered alone orin combination with cytotoxic factor(s) and/or cytokine(s), can be usedas a therapeutic.

The antibodies of the invention can be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the srcbiomarker-encoding polynucleotides and polypeptides of the presentinvention can be useful as cell specific marker(s), or morespecifically, as cellular marker(s) that are differentially expressed atvarious stages of differentiation and/or maturation of particular celltypes. Monoclonal antibodies directed against a specific epitope, orcombination of epitopes, allow for the screening of cellular populationsexpressing the marker. Various techniques utilizing monoclonalantibodies can be employed to screen for cellular populations expressingthe marker(s), including magnetic separation using antibody-coatedmagnetic beads, “panning” with antibody(ies) attached to a solid matrix(i.e., tissue culture plate), and flow cytometry (See, e.g., U.S. Pat.No. 5,985,660; and Morrison et al., 1999, Cell, 96:737-749).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Antibodies according to this invention can be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include, but are not limited to, competitive and non-competitiveassay systems using techniques such as BIAcore analysis, FACS(Fluorescence Activated Cell Sorter) analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assays), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known and practiced in the art (see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety). Nonlimiting, exemplary immunoassays are describedbriefly below.

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (i.e., 1% NP40 or TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodiumphosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphataseand/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodiumvanadate); adding the antibody of interest to the cell lysate;incubating for a period of time (e.g., 1 to 4 hours) at 4° C.; addingprotein A and/or protein G sepharose beads to the cell lysate;incubating for about 60 minutes or more at 4° C.; washing the beads inlysis buffer; and resuspending the beads in SDS/sample buffer. Theability of the antibody of interest to immunoprecipitate a particularantigen can be assessed by, for example, Western blot analysis. One ofskill in the art would be knowledgeable as to the parameters that can bemodified to increase the binding of the antibody to an antigen anddecrease the background (e.g., pre-clearing the cell lysate withsepharose beads). For further discussion regarding immunoprecipitationprotocols, see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, at10.16.1.

Western blot analysis generally comprises preparing protein samples;electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS PAGE depending on the molecular weight of the antigen);transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon; blocking the membrane inblocking solution (e.g., PBS with 3% BSA or nonfat milk); washing themembrane in washing buffer (e.g., PBS-Tween 20); blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer; washing the membrane in washing buffer; blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer; washing the membrane inwash buffer; and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding Western blot protocols, see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, at 10.8.1.

ELISAs comprise preparing antigen; coating the wells of a 96 wellmicrotiter plate with antigen; adding to the wells the antibody ofinterest conjugated to a detectable compound such as an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase);incubating for a period of time; and detecting the presence of theantigen. In ELISAs, the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundcan be added to the wells. Further, instead of coating the wells withantigen, the antibody can be first coated onto the well. In this case, asecond antibody conjugated to a detectable compound can be added to theantibody-coated wells following the addition of the antigen of interest.One of skill in the art would be knowledgeable as to the parameters thatcan be modified to increase the signal detected, as well as othervariations of ELISAs known in the art. For further discussion regardingELISAs, see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassayinvolving the incubation of labeled antigen (e.g., ³H or ¹²⁵I), or afragment or variant thereof, with the antibody of interest in thepresence of increasing amounts of labeled antigen, and the detection ofthe antibody bound to the labeled antigen. The affinity of the antibodyof interest for a src biomarker protein and the binding off rates can bedetermined from the data by Scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, the src biomarker protein is incubated with antibody of interestconjugated to a labeled compound (e.g., a compound labeled with ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody. This kind of competitive assay between two antibodies, mayalso be used to determine if two antibodies bind to the same ordifferent epitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of antibodies (including antibody fragmentsor variants thereof) to a src biomarker protein, or fragments of a srcbiomarker protein. Kinetic analysis comprises analyzing the binding anddissociation of antibodies from chips with immobilized src biomarkerprotein on the chip surface.

It is to be further understood that the above-described techniques forthe production, expression, isolation, and manipulation of antibodymolecules, for example, by recombinant techniques involving molecularbiology, as well as by other techniques related to the analysis ofpolynucleotides and polypeptides and proteins, are applicable to otherpolypeptide or peptide molecules of the invention as described herein,in particular, the src biomarker polypeptides or peptides themselves, asapplicable or warranted in accordance with the various embodiments ofthis invention.

The present invention also embraces a kit for determining or predictingdrug susceptibility or resistance by a patient having a disease,particularly a cancer or tumor, preferably, a colon cancer or tumor.Such kits are useful in a clinical setting for use in testing patient'sbiopsied tumor or cancer samples, for example, to determine or predictif the patient's tumor or cancer will be resistant or sensitive to agiven treatment or therapy with a drug, compound, chemotherapy agent, orbiological treatment agent. Provided in the kit are the predictor setcomprising those polynucleotides and polypeptides correlating withresistance and sensitivity to src or src family tyrosine kinasesmodulators in a particular biological system, particularly src kinaseinhibitors, and preferably comprising a microarray; and, in suitablecontainers, the modulator compounds for use in testing a cells frompatient tissue or patient samples for resistance/sensitivity; andinstructions for use. Such kits encompass predictor set comprising thosepolynucleotides and polypeptides correlating with resistance andsensitivity to modulators of protein tyrosine kinases including membersof the Src family of tyrosine kinases, for example, Src, Fgr, Fyn, Yes,Blk, Hck, Lck and Lyn, as well as other protein tyrosine kinases,including, Bcr-abl, Jak, PDGFR, c-kit and Ephr,

Also, as explained above, the kit is not limited to microarrays, but canencompass a variety of methods and systems by which the expression ofthe predictor/marker polynucleotides and polypeptides can be assayedand/or monitored, both at the level of mRNA and of protein, for example,via PCR assays, e.g., RT-PCR and immunoassay, such as ELISA. In kits forperforming PCR, or in situ hybridization, for example, nucleic acidprimers or probes from the sequences of one or more of the predictorpolynucleotides and polypeptides, such as those described in Tables 3-6and 13, are supplied, in addition to buffers and reagents as necessaryfor performing the method, and instructions for use. In kits forperforming immunoassays, e.g. ELISAs, immunoblotting assays, and thelike, antibodies, or bindable portions thereof, to the src biomarkerpolypeptides of the invention, or to antigenic or immunogenic peptidesthereof, are supplied, in addition to buffers and reagents as necessaryfor performing the method, and instructions for use.

In another embodiment, the present invention embraces the use of one ormore polynucleotides and polypeptides among those of the predictorpolynucleotides and polypeptides identified herein that can serve astargets for the development of drug therapies for disease treatment.Such targets may be particularly applicable to treatment of colondisease, such as colon cancers or tumors. Indeed, because thesepredictor polynucleotides and polypeptides are differently expressed insensitive and resistant cells, their expression pattern is correlatedwith relative intrinsic sensitivity of cells to treatment with compoundsthat interact with and inhibit src tyrosine kinases. Accordingly, thepolynucleotides and polypeptides highly expressed in resistant cells mayserve as targets for the development of drug therapies for the tumorswhich are resistant to src tyrosine kinase inhibitor compounds.

EXAMPLES

The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way. The Examples do not include detaileddescriptions for conventional methods employed, such as in theconstruction of vectors, the insertion of cDNA into such vectors, or theintroduction of the resulting vectors into the appropriate host. Suchmethods are well known to those skilled in the art and are described innumerous publications, for example, Sambrook, Fritsch, and Maniatis,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Laboratory Press, USA, (1989).

Example 1 Methods IC₅₀ Determination—In Vitro Cytotoxicity Assay

Src tyrosine kinase inhibitor compounds (described in WO 00/62778,published Oct. 26, 2000) were tested for cytotoxicity in vitro against apanel of thirty-one human colon cell lines available from the AmericanType Culture Collection, ATCC, except CX-1 and MIP, which were obtainedfrom academic investigators. Cytotoxicity was assessed in cells by theMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulphenyl)-2H-tetrazolium,inner salt) assay (T. L. Riss et al., 1992, Mol. Biol. Cell, 3 (Suppl.):184a).

To carry out the assays, the colon cells were plated at 4,000 cells/wellin 96 well microtiter plates and 24 hours later serial diluted drugswere added. The concentration range for the src compounds used in thecytotoxicity assay was from 5 μg/ml to 0.0016 μg/ml (roughly 10 μM to0.0032 μM). The cells were incubated at 37° C. for 72 hours at whichtime the tetrazolium dye, MTS (333 μg/ml final concentration), incombination with the electron coupling agent phenazine methosulfate (25μM final concentration), was added. A dehydrogenase enzyme in live cellsreduces the MTS to a form that absorbs light at 492 nM that can bequantified spectrophotometrically. The greater the absorbency thegreater the number of live cells. The results are expressed as an IC₅₀,which is the drug concentration required to inhibit cell proliferation(i.e. absorbance at 450 nM) to 50% of that of untreated control cells.The mean IC₅₀ and standard deviation (SD) from multiple tests for eachcell line were calculated.

Resistant/Sensitive Classification

For each compound, the IC₅₀ for each cell line was log-transformed tolog₁₀(IC₅₀), and the log₁₀(IC₅₀) values were then normalized across the31 colon cell lines. The cell lines with log₁₀(IC₅₀) below the meanlog₁₀(IC₅₀) of all 31 cell lines were defined as sensitive to thecompound, while those with log₁₀(IC₅₀) above the mean log₁₀(IC₅₀) wereconsidered to be resistant to the compound. The classification of thethirty-one colon cell lines was performed for all four of the src kinaseinhibitor compounds. (Table 2).Gene expression profiling

The colon cells were grown to 50-70% confluence, and RNA was isolatedusing the RNeasy™ kits (Qiagen, Valencia, Calif.). The quality of theRNA was assessed by measuring the 28s:18s ribosomal RNA ratio by usingan Agilent 2100 bioanalyzer (Agilent Technologies, Rockville, Md.). Theconcentration of total RNA was determined spectrophotometrically. 10 μgof total RNA from each cell line was used to prepare biotinylated probeaccording to the Affymetrix Manual (Affymetrix Genechip® TechnicalManual, 2000). Probes were hybridized to Affymetrix human genome U95Av2high density oligonucleotide arrays (Affymetrix, Santa Clara, Calif.).The arrays were then washed and stained using the GeneChip® Fluidicsstation according to the manufacture's instructions (AffymetrixGenechip® Technical Manual, 2000). The HG-U95Av2 array containsapproximately 12,000 probe sets which represent approximately 12,000human gene sequences and ESTs.

Preprocessing of Microarray Data

Scanned image files were visually inspected for artifacts and analyzedwith GeneChip® Expression Analysis software (Affymetrix, Santa Clara,Calif.). The “Absolute Call” (Affymetrix Genechip® Technical Manual,2000) which is used to determine whether a transcript is detected withinone sample, as well as the “Average Difference” (Affymetrix Genechip®Technical Manual, 2000), which serves as a relative indicator of thelevel of expression of a transcript, were calculated. The hybridizationintensity for each sample was scaled to 1,500 (Affymetrix Genechip®Technical Manual, 2000) in order to account for any minor differences inglobal chip intensity, so that the overall expression level for eachcell line was comparable. Affymetrix control sequences were removedprior to analysis.

Of a total of 12,558 represented polynucleotides and polypeptides on theHG-U9SAv2 array, 2079 represented polynucleotides and polypeptides werenot detected (Absent Call) across all of the thirty-one colon cell linesusing the Affymetrix GeneChip® Expression Analysis algorithm; theseundetected polynucleotides and polypeptides were excluded from furtheranalysis. The remaining data were transferred to the GeneClustersoftware (Whitehead Institute; T. R. Golub et al., 1999, Science,286:531-537). A threshold filter was applied to the gene expressionvalues of the remaining 10,479 represented polynucleotides andpolypeptides to remove negative gene expression values and to limit highgene expression values that were not likely to be in the linear range ofthe Affymetrix fluorescent scanner. The threshold filter converted allgene expression values that were negative, or below 100 units, to 100units, and all gene expression values that were above 40,000 units to40,000 units. All represented polynucleotides and polypeptides whosegene expression values were between 100 and 40,000 were not changed.

A second “variation filter” was then applied to the data set to findpolynucleotides and polypeptides that were likely to correlate withdifferent properties and features of the panel of thirty-one cell lines.The object of the second filter is to select those polynucleotides andpolypeptides whose expression pattern varies across the data set; a genethat does not vary can not provide information about differingproperties of the thirty-one cell line panel. For example, if there aretwo populations of cells within the data set, i.e., fast growing cellsand slow growing cells, then a gene whose expression is constant, orwhose expression does not change substantially, can not yieldinformation that would correlate to fast or slow cell growth.

The second variation filter was formulated to determine the expressionpattern of each gene across the thirty-one cell lines and findpolynucleotides and polypeptides that passed the following criteria:

-   -   1. The gene must show a three-fold change in absolute        expression, i.e., as depicted in the formula:        $\frac{{expression}\quad{value}\quad{in}\quad{any}\quad{give}\quad{cell}\quad{line}}{{expression}\quad{value}\quad{in}\quad{any}\quad{other}\quad{cell}\quad{line}} > {3\quad{or}} < 0.33$    -   2. In addition to 1, the three-fold change must represent an        absolute difference of 1000 expression units.    -   3. In addition, the criteria in #1 and #2 above must be met on        three independent occasions within the data set, i.e., Cell line        A/B, Cell line E/F and Cell line T/G. (The algorithm does not        use a single expression value for one cell line on multiple        occasions, i.e., Cell Line A/B, Cell line A/F and Cell line        B/F).

The second variation filter reduced the data set to 3008 polynucleotidesand polypeptides.

After the second variation filter was applied, each gene was normalizedto the mean across all the thirty-one colon cell samples (with the meanset to 0 and standard deviation set to 1) using the following formula:$\frac{\begin{matrix}{{{Expresssion}\quad{value}\quad{gene}\quad{``Z"}} -} \\{{mean}\quad{expresssion}\quad{value}\quad{of}\quad{gene}\quad{``Z"}\quad{in}\quad{the}\quad 31\quad{cell}\quad{lines}}\end{matrix}}{\begin{matrix}{{{Standard}\quad{deviation}\quad{of}\quad{expression}}\quad} \\{{{value}\quad{for}\quad{gene}\quad{``Z"}\quad{in}\quad{the}\quad 31\quad{cell}\quad{lines}}\quad}\end{matrix}}$

This normalized data set was used to select polynucleotides andpolypeptides which significantly correlated with the property ofsensitivity toward a drug class as described herein.

Example 2 PCR Expression Profiling

RNA quantification is performed using the Taqman® real-time-PCRfluorogenic assay. The Taqman® assay is one of the most precise methodsfor assaying the concentration of nucleic acid templates.

All cell lines are grown using standard cell culture conditions: RPMI1640 supplemented to contain 10% fetal bovine serum, 100 IU/mlpenicillin, 100 mg/ml streptomycin, 2 mM L-glutamine and 10 mM Hepes(all from GibcoBRL, Rockville, Md.). Eighty percent confluent cells arewashed twice with phosphate-buffered saline (GibcoBRL) and harvestedusing 0.25% trypsin (GibcoBRL). RNA is prepared using standard methods,preferably, employing the RNeasy Kit commercially available from Qiagen(Valencia, Calif.).

cDNA template for real-time PCR can be generated using the Superscript™First Strand Synthesis system for RT-PCR. Representative forward andreverse RT-PCT primers for each of the src biomarker polynucleotides andpolypeptides of the present invention are provided in Tables 13 herein.

SYBR Green real-time PCR reactions are prepared as follows: The reactionmix contains 20 ng first strand cDNA; 50 nM Forward Primer (one or moreprimers selected from SEQ ID NO:391 to 591); 50 nM Reverse Primer (oneor more primers selected from SEQ ID NO:592 to 792); 0.75×SYBR Green I(Sigma); 1×SYBR Green PCR Buffer (50 mMTris-HCl pH 8.3, 75 mM KCl); 10%DMSO; 3 nM MgCl₂; 300 μM each dATP, dGTP, dTTP, dCTP; 1 U Platinum® TaqDNA Polymerase High Fidelity (Cat# 11304029; Life Technologies;Rockville, Md.), 1:50 dilution; ROX (Life Technologies).

Real-time PCR is performed using an Applied Biosystems 5700 SequenceDetection System. Conditions are 95° C. for 10 minutes (denaturation andactivation of Platinum® Taq DNA Polymerase), 40 cycles of PCR (95° C.for 15 seconds, 60° C. for 1 minute). PCR products are analyzed foruniform melting using an analysis algorithm built into the 5700 SequenceDetection System.

cDNA quantification used in the normalization of template quantity isperformed using Taqman® technology. Taqman® reactions are prepared asfollows: The reaction mix comprises 20 ng first strand cDNA; 25 nMGAPDH-F3, Forward Primer, 250 nM GAPDH-R1 Reverse Primer; 200 nMGAPDH-PVIC Taqman® Probe (fluorescent dye labeled oligbnucleotideprimer); 1× Buffer A (Applied Biosystems); 5.5 mM MgCl₂; 300 μM DATP,dGTP, dTTP, dCTP; 1 U Amplitaq Gold (Applied Biosystems). GAPDH,D-glyceraldehyde-3-phosphate dehydrogenase, is used as a control tonormalize mRNA levels.

Real-time PCR is performed using an Applied Biosystems 7700 SequenceDetection System. Conditions are 95° C. for 10 minutes (denaturation andactivation of Amplitaq Gold), 40 cycles of PCR (95° C. for 15 seconds,60° C. for 1 minute).

The sequences for the GAPDH oligonucleotides used in the Taqman®reactions are as follows: GAPDH-F3: 5′-AGCCGAGCCACATCGCT-3′ (SEQ ID NO:793) GAPDH-R1: 5′-GTGACCAGGCGCCCAATAC-3′ (SEQ ID NO: 794) GAPDH-PVICCAAATCCGTTGACTCCGACCTTCACCTT-3′ (SEQ ID NO: 795) TAMRA.Taqman® Probe -VIC-5′-

The Sequence Detection System generates a Ct (threshold cycle) valuethat is used to calculate a concentration for each input cDNA template.cDNA levels for each gene of interest are normalized to GAPDH cDNAlevels to compensate for variations in total cDNA quantity in the inputsample. This is done by generating GAPDH Ct values for each cell line.Ct values for the gene of interest and GAPDH are inserted into amodified version of the δδCt equation (Applied Biosystems Prism® 7700Sequence Detection System User Bulletin #2), which is used to calculatea GAPDH normalized relative cDNA level for each specific cDNA. The δδCtequation is as follows: relative quantity of nucleic acidtemplate=2^(δδCt)=2^((δCta−δCtb)), where δCta=Ct target−Ct GAPDH, andδCtb=Ct reference−Ct GAPDH. (No reference cell line is used for thecalculation of relative quantity; δCtb is defined as 21).

Example 3 Production of an Antibody Directed Against Src BiomarkerPolypeptides

Anti-src biomarker polypeptide antibodies of the present invention canbe prepared by a variety of methods. As one example of anantibody-production method, cells expressing a polypeptide of thepresent invention are administered to an animal to induce the productionof sera containing polyclonal antibodies directed to the expressedpolypeptides. In a preferred method, the expressed protein is prepared,preferably isolated and purified, to render it substantially free ofnatural contaminants, using techniques commonly practiced in the art.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity for the expressed andisolated polypeptide.

In a most preferred method, the antibodies of the present invention aremonoclonal antibodies (or protein binding fragments thereof) and can beprepared using hybridoma technology as detailed hereinabove. Cellsexpressing the polypeptide can be cultured in any suitable tissueculture medium; however, it is preferable to culture cells in Earle'smodified Eagle's medium supplemented to contain 10% fetal bovine serum(inactivated at about 56° C.), and supplemented to contain about 10 g/lnonessential amino acids, about 1,000 U/ml penicillin, and about 100μg/ml streptomycin.

The splenocytes of immunized (and boosted) mice are extracted and fusedwith a suitable myeloma cell line. Any suitable myeloma cell line can beemployed in accordance with the present invention; however, it ispreferable to employ the parent myeloma cell line (SP2/0), availablefrom the ATCC. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al. (1981, Gastroenterology,80:225-232). The hybridoma cells obtained through such a selection arethen assayed to identify those cell clones that secrete antibodiescapable of binding to the polypeptide immunogen, or a portion thereof.

Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodythat binds to a second antibody. In accordance with this method, proteinspecific antibodies are used to immunize an animal, preferably a mouse.The splenocytes of such an immunized animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify clonesthat produce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce the formation of furtherprotein-specific antibodies.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known and practiced in the art. (See, e.g., forreview, Morrison, 1985, Science, 229:1202); Oi et al., 1986,BioTechniques, 4:214; Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchiet al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO8601533; Robinson et al., WO 8702671; Boulianne et al., 1984, Nature,312:643; and Neuberger et al., 1985, Nature, 314:268).

Example 4 Immunofluorescence Assays

The following immunofluorescence protocol may be used, for example, toverify src biomarker protein expression on cells, or, for example, tocheck for the presence of one or more antibodies that bind src biomarkerprotein expressed on the surface of cells. Briefly, Lab-Tek II chamberslides are coated overnight at 4° C. with 10 micrograms/milliliter(μg/ml) of bovine collagen Type II in DPBS containing calcium andmagnesium (DPBS++). The slides are then washed twice with cold DPBS++and seeded with 8000 CHO—CCR5 or CHO pC4 transfected cells in a totalvolume of 125 μl and incubated at 37° C. in the presence of 95%oxygen/5% carbon dioxide.

The culture medium is gently removed by aspiration and the adherentcells are washed twice with DPBS++ at ambient temperature. The slidesare blocked with DPBS++ containing 0.2% BSA (blocker) at 0-4° C. for onehour. The blocking solution is gently removed by aspiration, and 125 μlof antibody containing solution (an antibody containing solution may be,for example, a hybridoma culture supernatant which is usually usedundiluted, or serum/plasma which is usually diluted, e.g., a dilution ofabout 1/100 dilution). The slides are incubated for 1 hour at 0-4° C.Antibody solutions are then gently removed by aspiration and the cellsare washed 5 times with 400 μl of ice cold blocking solution. Next, 125μl of 1 μg/ml rhodamine labeled secondary antibody (e.g., anti-humanIgG) in blocker solution is added to the cells. Again, cells areincubated for 1 hour at 04° C.

The secondary antibody solution is then gently removed by aspiration andthe cells are washed 3 times with 400 μl of ice cold blocking solution,and 5 times with cold DPBS++. The cells are then fixed with 125 μl of3.7% formaldehyde in DPBS++for 15 minutes at ambient temperature.Thereafter, the cells are washed 5 times with 400 μl of DPBS++ atambient temperature. Finally, the cells are mounted in 50% aqueousglycerol, and viewed in a fluorescence microscope using rhodaminefilters.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Incorporated herein by reference in its entirety is a Sequence Listing,including SEQ ID NO:1 through SEQ ID NO:390, which include nucleic acidand amino acid sequences of the src biomarkers as presented in Tables3-6 herein. The Sequence Listing also contains representative primerpairs that may be used in RT-PCR assays for any of the predictorpolynucleotides and polypeptides of the present invention, including SEQID NO:391 through SEQ ID NO:792. TABLE 1 Mean IC50 Mean IC50 Mean IC50Mean IC50 Cell Lines (uM) +/− SD (uM) +/− SD (uM) +/− SD (uM) +/− SDWiDr 0.041 0.015 0.007 0.004 0.185 0.096 0.040 0.010 HT-29 0.042 0.0230.038 0.023 0.489 0.204 0.082 0.044 LoVo 0.055 0.041 0.005 0.002 1.7231.496 0.028 0.016 HCT-15 0.153 0.066 0.326 0.095 3.409 1.418 0.221 0.029CCD-18Co 0.215 0.247 0.028 0.043 1.955 1.611 0.099 0.111 Caco-2 0.2330.099 0.538 0.344 0.429 0.108 0.295 0.074 CCD-33Co 0.311 0.221 0.1240.178 1.068 0.745 0.241 0.079 LS174T 0.505 0.199 0.060 0.039 2.279 1.3540.356 0.191 SW1417 0.505 0.476 0.021 0.028 0.187 0.246 0.096 0.081 SW8373.318 1.276 3.985 3.055 0.460 0.263 2.110 1.588 DLD-1 4.506 1.002 2.1211.613 6.272 3.415 2.545 1.709 RKO-RM13 4.686 1.917 2.761 0.454 3.2960.749 5.552 1.647 MIP 6.548 2.311 5.822 1.294 10.565 1.557 6.097 3.398CX-1 7.801 3.677 7.925 3.886 2.517 1.225 7.691 1.287 HCT116-S542 8.7262.109 8.353 0.286 11.261 0.000 7.799 3.225 SK-CO-1 9.533 0.398 9.9120.000 9.038 4.446 8.900 0.000 Colo201 9.814 0.000 7.008 1.500 7.1201.755 6.972 2.832 Colo205 9.814 0.000 7.952 2.414 4.374 1.613 4.8912.474 Colo320DM 9.814 0.000 9.912 0.000 11.261 0.000 8.926 0.000 HCT1169.814 0.000 7.903 2.422 11.261 0.000 8.926 0.000 HCT-8 9.814 0.000 5.3623.142 7.743 3.991 7.312 1.974 SW403 9.814 0.000 0.123 0.171 0.206 0.1857.868 1.937 SW480 9.814 0.000 7.286 2.893 11.261 0.000 8.926 0.000 SW6209.814 0.000 7.622 0.827 11.261 0.000 8.926 0.000 T84 9.814 0.000 0.6820.492 0.715 0.238 4.994 3.406 Colo 320HSR 10.571 0.000 10.684 0.00011.261 0.000 9.524 0.000 LS1034 10.571 0.000 10.684 0.000 11.261 0.0009.524 0.000 LS180 10.571 0.000 1.890 0.620 11.261 0.000 2.558 0.544LS513 10.571 0.000 2.254 1.283 11.261 0.000 9.524 0.000 SW1116 10.5710.000 1.831 0.923 11.261 0.000 9.524 0.000 SW948 10.571 0.000 1.7561.007 11.261 0.000 9.524 0.000

TABLE 2 Cell Lines BMS-A BMS-B BMS-C BMS-D WiDr S S S S HT-29 S S S SLoVo S S S S CCD-18Co S S S S Caco-2 S S S S CCD-33Co S S S S LS174T S SS S SW1417 S S S S HCT-15 S S R S T84 R S S R SW403 R S S R SW837 R R SR CX-1 R R S R DLD-1 R R R R RKO-RM13 R R R R MIP R R R R HCT116-S542 RR R R SK-CO-1 R R R R Colo201 R R R R Colo205 R R R R Colo320DM R R R RHCT116 R R R R HCT-8 R R R R SW480 R R R R SW620 R R R R Colo 320HSR R RR R LS1034 R R R R LS180 R R R R LS513 R R R R SW1116 R R R R SW948 R RR R

TABLE 3 Common to all 4 BMS Highly compounds SEQ ID Expressed (BMS-A,BMS- Common to Common to SEQ ID NO: of Cells (Sensitive B, BMS-C, BMS-BBMS-C Genbank NO: of Amino Gene # or Resistent) BMS-D) Compound CompoundAccession # UniGene Title DNA Acid 1 Sensitive cells yes AB014558cryptochrome 2 (photolyase-like) 1 202 2 Sensitive cells yes NM_006979HLA class II region expressed gene 2 203 KE4 3 Sensitive cells yesM22489 bone morphogenetic protein 2 3 204 4 Sensitive cells yes AB023194KIAA0977 protein 4 205 5 Sensitive cells yes U03688 cytochrome P450,subfamily I 5 206 (dioxin-inducible), polypeptide 1 (glaucoma 3, primaryinfantile) 6 Sensitive cells yes M88458 KDEL (Lys-Asp-Glu-Leu) 6 207endoplasmic reticulum protein retention receptor 2 7 Sensitive cells yesL13463 regulator of G-protein signalling 2, 7 208 24 kD 8 Sensitivecells yes U21551 branched chain aminotransferase 1, 8 209 cytosolic 9Sensitive cells yes AF000560 Homo sapiens TTF-I interacting 9 210peptide 20 mRNA, partial cds 10 Sensitive cells AF102265N-acetylglucosamine-phosphate 10 211 mutase 11 Sensitive cells yesX06272 signal recognition particle receptor 11 212 (‘docking protein’)12 Sensitive cells yes L40802 hydroxysteroid (17-beta) 12 213dehydrogenase 2 13 Sensitive cells yes X13916 low densitylipoprotein-related 13 214 protein 1 (alpha-2-macroglobulin receptor) 14Sensitive cells yes AF009674 axin 14 215 15 Sensitive cells yes M73077glucocorticoid receptor DNA 15 216 binding factor 1 16 Sensitive cellsyes U15655 Ets2 repressor factor 16 217 17 Sensitive cells AB014520KIAA0620 protein 17 218 18 Sensitive cells yes M58603 nuclear factor ofkappa light 18 219 polypeptide gene enhancer in B- cells I (p105) 19Sensitive cells X76104 death-associated protein kinase 1 19 220 20Sensitive cells yes A1659108 Homo sapiens, clone 20 N/A IMAGE: 3908182,mRNA, partial cds 21 Sensitive cells yes U72649 BTG family, member 2 21221 22 Sensitive cells yes M64571 microtubule-associated protein 4 22222 23 Sensitive cells yes X77909 nuclear factor of kappa light 23 223polypeptide gene enhancer is B- cells inhibitor-like 1 24 Sensitivecells M34064 cadherin 2, type 1, N-cadherin 24 224 (neuronal) 25Sensitive cells AL050345 chromosome 22 open reading frame 2 25 225 26Sensitive cells yes AB006622 KIAA0284 protein 26 226 27 Sensitive cellsyes AB029027 KIAA1104 protein 27 227 28 Sensitive cells yes U51903 IQmotif containing GTPase 28 228 activating protein 2 29 Sensitive cellsAF041259 zinc finger protein 217 29 229 30 Sensitive cells yes AB026891solute carrier family 7, (cationic 30 230 amino acid transporter, y+system) member 11 31 Sensitive cells AB007960 SH3-domain, GRB2-like, 31231 endophilin B1 32 Sensitive cells D63390 platelet-activating factor32 232 acetylhydrolase, isoform Ib, beta subunit (30 kD) 33 Sensitivecells L10678 profilin 2 33 233 34 Sensitive cells yes X60708dipeptidylpeptidase IV (CD26, 34 234 adenosine deaminase complexingprotein 2) 35 Sensitive cells Y15521 acetylserotonin O- 35 235methyltransferase-like 36 Sensitive cells yes AI038821 v-Ha-ras Harveyrat sarcoma viral 36 236 oncogene homolog 37 Sensitive cells X84740ligase III, DNA, ATP-dependent 37 237 38 Sensitive cells M23115 ATPase,Ca++ transporting, cardiac 38 238 muscle, slow twitch 2 39 Sensitivecells NM017432 prostate tumor over expressed gene 1 39 239 40 Sensitivecells Y12781 transducin (beta)-like 1 40 240 41 Sensitive cells yesK03498 Homo sapiens endogenous 41 241 retrovirus HERV-K104 long terminalrepeat, complete sequence; and Gag protein (gag) end envelope protein(env) polynucleotides and polypeptides, complete cds 42 Sensitive cellsAF030335 purinergic receptor P2Y, G-protein 42 242 coupled, 11 43Sensitive cells X93209 nardilysin (N-arginine dibasic 43 243 convertase)44 Sensitive cells AF068744 double homeobox, 2 44 244 45 Sensitive cellsAF072247 methyl-CpG binding domain 45 245 protein 3 46 Sensitive cellsyes U41344 proline arginine-rich end leucine- 46 246 rich repeat protein47 Sensitive cells yes D13413 heterogeneous nuclear 47 247ribonucleoprotein U (scaffold attachment factor A) 48 Sensitive cellsyes M69023 tetraspan 3 48 248 49 Sensitive cells J04599 biglycan 49 24950 Sensitive cells U79267 protein phosphatase 4, regulatory 50 250subunit 1 51 Sensitive cells yes AF155654 Human putative ribosomalprotein 51 251 S1 mRNA 52 Sensitive cells X12794 nuclear receptorsubfamily 2, group 52 252 F, member 6 53 Sensitive cells U51166thymine-DNA glycosylase 53 253 54 Sensitive cells yes L07261 adducin 1(alpha) 54 254 55 Sensitive cells U97188 IGF-II mRNA-binding protein 355 255 56 Sensitive cells yes L37033 FK506-binding protein 8 (38 kD) 56256 57 Sensitive cells yes Y09846 SHC (Src homology 2 domain- 57 257containing) transforming protein 1 58 Sensitive cells yes AF093420hsp70-interacting protein 58 258 59 Sensitive cells yes U19775mitogen-activated protein kinase 14 59 259 60 Sensitive cells J04027ATPase, Ca++ transporting, plasma 60 260 membrane 1 61 Resistant cellsyes Y18483 solute carrier family 7 (cationic 61 261 amino acidtransporter, y+ system), member 8 62 Resistant cells yes U57352amiloride-sensitive cation channel 62 262 1, neuronal (degenerin) 63Resistant cells yes U34994 protein kinase, DNA-activated, 63 263catalytic polypeptide 64 Resistant cells yes X79067 zinc finger protein36, C3H type- 64 264 like 1 65 Resistant cells yes AB011535 FAT tumorsuppressor (Drosophila) 65 265 homolog 2 66 Resistant cells yes U90902Human clone 23612 mRNA 66 N/A sequence 67 Resistant cells AB009282cytochrome b5 outer mitochondrial 67 266 membrane precursor 68 Resistantcells yes AJ001685 killer cell lectin-like receptor 68 267 subfamily C,member 3 69 Resistant cells yes S37730 insulin-like growth factorbinding 69 268 protein 2 (36 kD) 70 Resistant cells yes U37518 tumornecrosis factor (ligand) 70 269 superfamily, member 10 71 Resistantcells yes AC005329 NADH dehydrogenase (ubiquinone) 71 270 Fe-S protein 7(20 kD) (NADH- coenzyme Q reductase) 72 Resistant cells yes AB009426apolipoprotein B mRNA editing 72 271 enzyme, catalytic polypeptide 1 73Resistant cells yes X70340 transforming growth factor, alpha 73 272 74Resistant cells yes U81561 protein tyrosine phosphatase, 74 273 receptortype, N polypeptide 2 75 Resistant cells yes X70040 macrophagestimulating 1 receptor 75 274 (c-met-related lyrosine kinase) 76Resistant cells yes AB000449 vaccinia related kinase 1 76 275 77Resistant cells yes D87119 GS3955 protein 77 276 78 Resistant cells yesX06745 polymerase (DNA directed), alpha 78 277 79 Resistant cells yesX78817 Rho GTPase activating protein 4 79 278 80 Resistant cells yesAF070530 hypothetical protein, clone 24751 80 279 81 Resistant cells yesL43821 enhancer of filamentation 1 (cas- 81 280 like docking;Crk-associated substrate related) 82 Resistant cells yes AF007156KIAA0751 gene product 82 281 83 Resistant cells yes AB014566 KIAA0666protein 83 282 84 Resistant cells yes U71364 serine (or cysteine)proteinase 84 283 inhibitor, clade B (ovalbumin), member 9 85 Resistantcells U93305 Homo sapiens A4 differentiation- 85 284 dependent protein(A4), triple LIM domain protein (LMO6), and synaptophysin (SYP)polynucleotides and polypeptides, complete cds; and calcium channelalpha-1 subunit (CACNA1F) gene, partial cds 86 Resistant cells yesAB006626 histone deacetylase 4 86 285 87 Resistant cells yes M31682inhibin, beta B (activin AB beta 87 286 polypeptide) 88 Resistant cellsyes AF031824 cystain F (leukocystatin) 88 287 89 Resistant cells yesAF035299 docking protein 1, 62 kD 89 288 (downstream of tyrosinekinase 1) 90 Resistant cells X82207 ARP1 (actin-related protein 1, 90289 yeast) homolog B (centractin beta) 91 Resistant cells yes U84570chromosome 21 open reading frame 2 91 290 92 Resistant cells AA873266pyruvate dehydrogenase kinase, 92 291 isoenzyme 3 93 Resistant cells yesX90976 runt-related transcription factor 1 93 292 (acute mycloidleukemin 1; aml1 oncogene) 94 Resistant cells yes D89377 msh(Drosophila) homeo box 94 293 homolog 2 95 Resistant cells M57730ephrin-A1 95 294 96 Resistant cells yes U68111 protein phosphatase 1,regulatory 96 295 (inhibitor) subunit 2 97 Resistant cells yes L07540replication factor C (activator 1) 5 97 296 (36.5 kD) 98 Resistant cellsM65066 protein kinase, cAMP-dependent, 98 297 regulatory, type I, beta99 Resistant cells yes M34182 protein kinase, cAMP-dependent, 99 299catalytic, gamma 100 Resistant cells yes L34059 cadherin 4, type 1,R-cadherin 100 299 (retinal) 101 Resistant cells L25665 guaminenucleotide binding protein- 101 300 like 1 102 Resistant cells yesAL050290 spermidine/spermine N1- 102 301 acetyltransferase 103 Resistantcells X67325 interferon, alpha-inducible protein 103 302 27 104Resistant cells yes AA595596 ADP-ribosyltransferase (NAD+; 104 N/Apoly(ADP-ribose) polymerase)-like 2 105 Resistant cells yes AF003837jagged 1 (Alagille syndrome) 105 303 106 Resistant cells yes M87339replication factor C (activator 1) 4 106 304 (37 kD) 107 Resistant cellsAI813532 tumor necrosis factor receptor 107 N/A superfamily, member 1B108 Resistant cells yes AB018306 KIAA0763 gene product 108 305 109Resistant cells yes AI761647 Home saplens clone IMAGE 21721 109 N/A 110Resistant cells yes X80507 Yes-associated protein 1, 65 kDa 110 306 111Resistant cells Y16241 nebulette 111 307 112 Resistant cells D67031adducin 3 (gamma) 112 308 113 Resistant cells J05581 mucin 1,transmembrane 113 309 114 Resistant cells yes U19718microfibrillar-associated protein 2 114 310 115 Resistant cells yesU52840 sema domain, seven 115 311 thrombospondin repeats (type 1 andtype 1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5A 116 Resistant cells yes AB014557 KIAA0657 protein 116312 117 Resistant cells yes AF038172 hypothetical protein FLJ11149 117N/A 118 Resistant cells AB014529 A kinase (PRKA) anchor protein 11 118313 119 Resistant cells yes U65676 Hermansky-Pudlak syndrome 119 314 120Resistant cells yes U14971 ribosomal protein S9 120 315 121 Resistantcells yes X74331 primase, polypeptide 2A (58 kD) 121 316 122 Resistantcells yes D16815 nuclear receptor subfamily 1, group 122 317 D, member 2123 Resistant cells yes M14333 Homo sapiens cDNA FLJ32137 fis, 123 318clone PEBLM2000479, highly similar to PROTO-ONCOGENE TYROSINE-PROTEINKINASE FYN (EC 2.7.1.112)

TABLE 4 Common to all 4 BMS Highly compounds SEQ ID Expressed (BMS-A,BMS- Common to Common to SEQ ID NO: of Cells (Sensitive B, BMS-C, BMS-BBMS-C Genbank NO: of Amino Gene # or Resistent) BMS-D) Compound CompoundAccession # UniGene Title DNA Acid 1 Sensitive cells yes M22489 bonemorphogenetic protein 2 3 204 2 Sensitive cells yes AB023194 KIAA0977protein 4 205 3 Sensitive cells yes AF009674 axin 14 215 4 Sensitivecells yes NM_006979 HLA class II region expressed gene 2 203 KE4 5Sensitive cells yes M28668 cystic fibrosis transmembrane 124 319conductance regulator, ATP- binding cassette (sub-family C, member 7) 6Sensitive cells yes AF000560 Homo sapiens TTF-I interacting 9 210peptide 20 mRNA, partial cds 7 Sensitive cells yes M88458 KDEL(Lys-Asp-Glu-Leu) 6 207 endoplasmic reticulum protein retention receptor2 8 Sensitive cells yes AB006622 KIAA0284 protein 26 226 9 Sensitivecells yes AB014558 cryptochrome 2 (photolyase-like) 1 202 10 Sensitivecells yes M58603 nuclear factor of kappa light 18 219 polypeptide geneenhancer in B- cells 1 (p105) 11 Sensitive cells yes W29065 ESTs, Weaklysimilar to A28996 125 N/A proline-rich protein M14 precursor - mouse [M.musculus] 12 Sensitive cells yes U03688 cytochrome P450, subfamily I 5206 (dioxin-inducible), polypeptide 1 (glaucoma 3, primary infantile) 13Sensitive cells yes K03498 Homo sapiens endogenous 41 241 retrovirusHERV-K104 long terminal repeat, complete sequence; and Gag protein (gag)and envelope protein (env) polynucleotides and polypeptides, completecds 14 Sensitive cells yes M73077 glucocorticoid receptor DNA 15 216binding factor 1 15 Sensitive cells yes L40802 hydroxysteroid (17-beta)12 213 dehydrogenase 2 16 Sensitive cells yes AL050025 ESTs 126 320 17Sensitive cells yes AB026891 solute carrier family 7, (cationic 30 230amino acid transporter; y+ system) member 11 18 Sensitive cells yesAF000561 HIV-1 inducer of short transcripts 127 321 binding protein;lymphoma related factor 19 Sensitive cells yes U15655 Ets2 repressorfactor 16 217 20 Sensitive cells yes U41344 proline arginine-rich endleucine- 46 246 rich repeat protein 21 Sensitive cells yes M69023tetraspan 3 47 247 22 Sensitive cells yes AB008515 retinotic acidrepressible protein 48 248 23 Sensitive cells AJ011736 GRB2-relatedadaptor protein 2 129 323 24 Sensitive cells yes U72649 BTG family,member 2 21 221 25 Sensitive cells yes U21551 branched chainaminotransferase 1, 8 209 cytosolic 26 Sensitive cells yes D13413heterogeneous nuclear 47 247 ribonucleoprotein U (scaffold attachmentfactor A) 27 Sensitive cells yes U19775 mitogen-activated protein kinase14 59 259 28 Sensitive cells J00277 Human (genomic clones lambda- 130324 [SK2-T2, HS578T]; cDNA clones RS-

3, 4, 29 Sensitive cells yes X77909 c-Ha-ras1 proto-oncogene 23 223 30Sensitive cells yes Y10055 phospboinositide-3-kinase, 131 325 catalytic,delta polypeptide 31 Sensitive cells yes AB029027 KIAA1104 protein 27227 32 Sensitive cells yes X60708 dipeptidylpeptidase IV (CD26, 34 234adenosine deaminase complexing protein 2) 33 Sensitive cells yes X13916low density lipoprotein-related 13 214 protein 1 (alpha-2-macroglobulinreceptor) 34 Sensitive cells yes L37033 FK506-binding protein 8 (38 kD)56 256 35 Sensitive cells yes AF155654 Human putative ribosomal protein51 251 S1 mRNA 36 Sensitive cells yes L13463 regulator of G-proteinsignalling 2, 7 208 24 kD 37 Sensitive cells yes AI659108 Homo sapiens,clone 20 N/A IMAGE: 3908182, mRNA, partial cds 38 Sensitive cells W26652PTEN induced putative kinase 1 132 N/A 39 Sensitive cells yes U51903 IQmotif containing GTPase 28 228 activating protein 2 40 Sensitive cellsyes M11717 heat shock 70 kD protein 1A 133 326 41 Sensitive cells yesL32976 mitogen-activated protein kinase 134 327 kinase kinase 11 42Sensitive cells yes L07261 adducin 1 (alpha) 54 254 43 Sensitive cellsM29893 v-ral simian leukemia viral 135 328 oncogene homolog A (rasrelated) 44 Sensitive cells S70154 acetyl-Coenzyme A 136 329acetyltransferase 2 (acetoacetyl Coenzyme A thiolase) 45 Sensitive cellsyes D83542 cadherin 15, M-cadherin 137 330 (myotubule) 46 Sensitivecells Z74615 collagen, type I, alpha 1 138 331 47 Sensitive cells yesM96684 purine-rich element binding protein A 139 332 48 Sensitive cellsyes AF093420 hsp70-interacting protein 58 258 49 Sensitive cells yesD12763 interleukin 1 receptor-like 1 140 333 50 Sensitive cells S67070heat shock 27 kD protein 2 141 334 51 Sensitive cells yes M64571microtubule-associated protein 4 22 222 52 Sensitive cells yes Y09846SHC (Src homology 2 domain- 57 257 containing) transforming protein 1 53Sensitive cells X66435 3-hydroxy-3-methylglutaryl- 142 335 Coenzyme Asynthase 1 (soluble) 54 Sensitive cells U25138 potassium largeconductance 143 336 calcium-activated channel, subfamily M, beta member1 55 Sensitive cells yes D85131 MYC-associated zinc finger protein 144337 (purine-binding transcription factor) 56 Resistant cells yesAC005329 NADH dehydrogenase (ubiquinone) 71 270 Fe-S protein 7 (20 kD)(NADH- coenzyme Q reductase) 57 Resistant cells yes AJ001685 killer celllectin-like receptor 68 267 subfamily C, member 3 58 Resistant cells yesU90902 Human clone 23612 mRNA 66 N/A sequence 59 Resistant cells yesX70340 transforming growth factor, alpha 73 272 60 Resistant cells yesX79067 zinc finger protein 36, C3H type- 64 264 like 1 61 Resistantcells yes Y18483 solute carrier family 7 (cationic 61 261 amino acidtransporter, y+ system), member 8 62 Resistant cells yes U57352amiloride-sensitive cation channel 62 262 1, neuronal (degenerin) 63Resistant cells yes S37730 insulin-like growth factor binding 69 268protein 2 (36 kD) 64 Resistant cells yes D87119 GS3955 protein 77 276 65Resistant cells yes M36089 X-ray repair complementing 145 338 defectiverepair in Chinese hamaster cells 1 66 Resistant cells yes U34994 proteinkinase, DNA-activated, 63 263 catalytic polypeptide 67 Resistant cellsyes AC004472 Homo sapiens chromosome 9, P1 146 339 clone 11659 68Resistant cells yes AB009010 ubiquitin C 147 340 69 Resistant cellsD26158 ELAV (embryonic lethal, abnormal 148 341 vision, Drosophila)-like3 (Hu antigen C) 70 Resistant cells yes AF007156 KIAA0751 gene product82 281 71 Resistant cells yes AF031824 cystatin F (leukocystatin) 88 28772 Resistant cells yes AA595596 ADP-ribosyltransferase (NAD+; 104 N/Apoly(ADP-ribose) polymerase)-like 2 73 Resistant cells yes AL035307 H.sapiens gene from PAC 42616 149 342 74 Resistant cells yes AB000449vaccinia related kinase 1 76 275 75 Resistant cells yes AF070530hypothetical protein, clone 24751 80 279 76 Resistant cells yes AB011535FAT tumor suppressor (Drosophila) 65 265 homolog 2 77 Resistant cellsyes AB014566 KIAA0666 protein 83 282 78 Resistant cells yes U81561protein tyrosine phosphatase, 74 273 receptor type, N polypeptide 2 79Resistant cells yes M31682 inhibin, beta B (activin AB beta 87 286polypeptide) 80 Resistant cells yes X90976 runt-related transcriptionfactor 1 93 292 (acute myeloid leukemia 1; aml1 oncogene) 81 Resistantcells yes X06745 polymerase (DNA directed), alpha 78 277 82 Resistantcells yes AL043470 hypothetical protein FLJ10335 150 N/A 83 Resistantcells yes U84570 chromosome 21 open reading frame 2 91 290 84 Resistantcells yes L34059 cadherin 4, type 1, R-cadherin 100 299 (retinal) 85Resistant cells yes U19718 microfibrillar-associated protein 2 114 31086 Resistant cells yes U52840 sema domain, seven 115 311 thrombospondinrepeats (type 1 and type 1-like), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 5A 87 Resistant cells yes L07540replication factor C (activator 1) 5 97 296 (36.5 KD) 88 Resistant cellsyes U67733 phosphodiesterase 2A, cGMP- 151 343 stimulated 89 Resistantcells D28118 zinc finger protein 161 152 344 90 Resistant cells yesAB020661 KIAA0854 protein 153 345 91 Resistant cells yes U68111 proteinphosphatase 1, regulatory 96 295 (inhibitor) subunit 2 92 Resistantcells W72186 S100 calcium-binding protein A4 154 346 (calcium protien,calvasculin, metastasin, murine placental homolog) 93 Resistant cellsyes L43821 enhancer of filamentation 1 (cns- 81 280 like docking;Crk-associated substrate related) 94 Resistant cells U97067 catenin(cadherin-associated 155 347 protein), alpha-like 1 95 Resistant cellsyes AF003837 jagged 1 (Alagille syndrome) 105 303 96 Resistant cells yesAF038172 hypothetical protein FLJ11149 117 N/A 97 Resistant cellsAF0361261 hypothetical PRO2032 156 348 98 Resistant cells yes AB018306KIAA0763 gene product 108 305 99 Resistant cells yes M14333 Homo sapienscDNA FLJ32137 fis, 123 318 clone PEBLM2000479, highly similar toPROTO-ONCOGENE TYROSINE-PROTEIN KINASE FYN (EC 2.7.1.112) 100 Resistantcells yes AB007870 KIAA0410 gene product 157 349 101 Resistant cells yesAB014557 KIAA0657 protein 116 312 102 Resistant cells yes AB009426apolipoprotein B mRNA editing 72 271 enzyme, catalytic polypeptide 1 103Resistant cells yes U37518 tumor necrosis factor (ligand) 70 269superfamily, member 10 104 Resistant cells yes X78817 Rho GTPaseactivating protein 4 79 278 105 Resistant cells yes AB006626 histonedeacetylase 4 86 285 106 Resistant cells AB014519 Rho-associated,coiled-coil 158 350 containing protein kinase 2 107 Resistant cells yesAL050290 spermidine/spermine N1- 102 301 acetyltransferase 108 Resistantcells yes D89377 msh (Drosophila) homeo box 94 293 homolog 2 109Resistant cells yes X74331 primase, polypeptide 2A (58 kD) 121 316 110Resistant cells yes AF035299 docking protein 1, 62 kD 89 288 (downstreamof tyrosine kinase 1) 111 Resistant cells yes X70040 macrophagestimulating 1 receptor 75 274 (c-met-related tyrosine kinase) 112Resistant cells yes U14971 ribosomal protein S9 120 315 113 Resistantcells yes U65676 Hermansky-Pudlak syndrome 119 314 114 Resistant cellsAB011123 KIAA0551 protein 159 351 115 Resistant cells yes M34182 proteinkinase, cAMP-dependent, 99 298 catalytic, gamma 116 Resistant cells yesD16815 nuclear receptor subfamily 1, group 122 317 D, member 2 117Resistant cells yes AI761647 Homo sapiens clone IMAGE 21721 109 N/A 118Resistant cells U09578 mitogen-activated protein kinase- 160 352activated protein kinase 3 119 Resistant cells yes M87339 replicationfactor C (activator 1) 4 106 304 (37 kD)

TABLE 5 Common to all 4 BMS Highly compounds Common to SEQ ID Expressed(BMS-A, BMS- BMS-A/ Common to SEQ ID NO: of Cells (Sensitive B, BMS-C,BMS-D BMS-B Genbank NO: of Amino Gene # or Resistent) BMS-D) CompoundCompound Accession # UniGene Title DNA Acid 1 Sensitive cells yes M22489bone morphogenetic protein 2 3 204 2 Sensitive cells yes AF009674 axin14 215 3 Sensitive cells yes D12763 interleukin 1 receptor-like 1 140333 4 Sensitive cells yes AF000560 Homo sapiens TTF-I interacting 9 210peptide 20 mRNA, partial cds 5 Sensitive cells yes AB014558 cryptochrome2 (photolyase-like) 1 202 6 Sensitive cells yes M28668 cystic fibrosistransmembrane 124 319 conductance regulator, ATP- binding cassette(sub-family C, member 7) 7 Sensitive cells yes M88458 KDEL(Lys-Asp-Glu-Leu) 6 207 endoplasmic reticulum protein retention receptor2 8 Sensitive cells Y17711 calcium binding atopy-related 161 353autoantigen 1 9 Sensitive cells yes M69023 tetraspan 3 48 248 10Sensitive cells L13972 sialyltransferase 4A (beta- 162 354 galactosidasealpha-2,3- sialytransferase) 11 Sensitive cells yes AB006622 KIAA0284protein 26 226 12 Sensitive cells AF055009 old astrocyte specificallyinduced 163 355 substance 13 Sensitive cells yes X06272 signalrecognition particle receptor 11 212 (‘docking protein’) 14 Sensitivecells yes W29065 ESTs, Weakly similar to A28996 125 N/A proline-richprotein M14 precursor - mouse [M. musculus] 15 Sensitive cells yesAB023194 KIAA0977 protein 4 205 16 Sensitive cells AF007155 Homo sapiensclone 23763 164 356 unknown mRNA, partial cds 17 Sensitive cells yesU19775 mitogen-activated protein kinase 14 59 259 18 Sensitive cells yesU03688 cytochrome P450, subfamily I 5 206 (dioxin-inducible),polypeptide 1 (glaucoma 3, primary infantile) 19 Sensitive cellsAB018324 KIAA0781 protein 165 357 20 Sensitive cells yes AF000561 HIV-1inducer of short transcripts 127 321 binding protein; lymphoma relatedfactor 21 Sensitive cells AB023154 KIAA0937 protein 166 358 22 Sensitivecells X78992 zinc finger protein 36, C3H type- 167 359 like 2 23Sensitive cells yes L40802 hydroxysteroid (17-beta) 12 213 dehydrogenase2 24 Sensitive cells yes M73077 glucocorticoid receptor DNA 15 216binding factor 1 25 Sensitive cells yes AF155654 Human putativeribosomal protein 51 251 S1 mRNA 26 Sensitive cells yes U15655 Ets2repressor factor 16 217 27 Sensitive cells M60299 collagen, type II,alpha 1 (primary 168 360 osteoarthritis, spondyloepiphyseal dysplasia,congenital) 28 Sensitive cells yes AL050025 ESTs 126 320 29 Sensitivecells M80482 paired basic amino acid cleaving 169 361 system 4 30Sensitive cells yes X60708 dipeptidylpeptidase IV (CD26, 34 234adenosine deaminase complexing protein 2) 31 Sensitive cells U47025ESTs, Moderately similar to 170 362 1701409A glycogen phosphorylase [H.sapiens] 32 Sensitive cells yes Y10055 phosphoinositide-3-kinase, 131325 catalytic, delta polypeptide 33 Sensitive cells yes L13463 regulatorof G-protein signalling 2, 7 208 24 kD 34 Sensitive cells Y10032serum/glucocorticoid regulated 171 363 kinase 35 Sensitive cells yesX77909 nuclear factor of kappa light 23 223 polypeptide gene enhancer inB- cells Inhibitor-like 1 36 Sensitive cells yes U51903 IQ motifcontaining GTPase 28 228 activating protein 2 37 Sensitive cells U57057coronin, actin-binding protein, 2A 172 364 38 Sensitive cells yes K03498Homo sapiens endogenous 41 241 retrovirus HERV-K104 long terminalrepeat, complete sequence; and Gag protein (gag) and envelope protein(env) polynucleotides and polypeptides 39 Sensitive cells X90392deoxyribonuclease I-like 1 173 365 40 Sensitive cells yes L32976mitogen-activated protein kinase 134 327 kinase kinase 11 41 Sensitivecells yes M96684 purine-rich element binding protein A 139 332 42Sensitive cells yes D83542 cadherin 15, M-cadherin 137 330 (myotubule)43 Sensitive cells yes AB026891 solute carrier family 7, (cationic 30230 amino acid transporter, y+ system) member 11 44 Sensitive cellsAA418437 chromosome 1 open reading frame 174 N/A 27 45 Sensitive cellsyes M11717 heat shock 70 kD protein 1A 133 326 46 Sensitive cells yesAI659108 Homo sapiens, clone 20 N/A IMAGE: 3908182, mRNA, partial cds 47Sensitive cells yes L07261 adducin 1 (alpha) 54 254 48 Sensitive cellsyes AF093420 hsp70-interacting protein 58 258 49 Sensitive cells yesD13413 heterogeneous nuclear 47 247 ribonucleoprotein U (scaffoldattachment factor A) 50 Sensitive cells yes AB008515 retinoic acidrepressible protein 128 322 51 Sensitive cells X84373 nuclear receptorinteracting protein 1 175 366 52 Sensitive cells XM_212189 Homo sapiensglutamate receptor, 176 367 ionotropic, N-methyl D-asparate- associatedprotein 1 (glutamate binding) (GRINA), mRNA 53 Sensitive cells yesU72649 D-asparate-associated protein 1 21 221 (glutamate binding)(GRINA), mRNA 54 Sensitive cells yes NM_006979 HLA class II regionexpressed gene 2 203 KE4 55 Sensitive cells yes M58603 nuclear factor ofkappa light 18 219 polypeptide gene enhancer in B- cells 1 (p105) 56Sensitive cells AF109134 opioid growth factor receptor 177 368 57Sensitive cells yes L37033 FK506-binding protein 8 (38 kD) 56 256 58Sensitive cells M64788 RAP1, GTPase activating protein 1 178 369 59Sensitive cells U43368 vascular endothelial growth factor B 179 370 60Sensitive cells yes AB029027 KIAA1104 protein 27 227 61 Sensitive cellsX13293 v-myb avian myeloblastosis viral 180 371 oncogene homolog-like 262 Sensitive cells yes D85131 MYC-associated zinc finger protein 144 337(purine-binding transcription factor) 63 Sensitive cells AB014511ATPase, Class II, type 9A 181 372 64 Sensitive cells yes X13916 lowdensity lipoprotein-related 13 214 protein 1 (alpha-2-macroglobulinreceptor) 65 Sensitive cells yes Y09846 SHC (Src homology 2 domain- 57257 containing) transforming protein 1 66 Resistant cells yes AC004472Homo sapiens chromosome 9, P1 146 339 clone 11659 67 Resistant cells yesU90902 Human clone 23612 mRNA 66 N/A sequence 68 Resistant cellsAB014585 KIAA0685 gene product 182 373 69 Resistant cells yes X06745polymerase (DNA directed), alpha 78 277 70 Resistant cells AB011114KIAA0542 gene product 183 374 71 Resistant cells yes AL035307 H. sapiensgene from PAC 42616 149 342 72 Resistant cells yes S37730 insulin-likegrowth factor binding 69 268 protein 2 (36 kD) 73 Resistant cells yesM36089 X-ray repair complementing 145 338 defective repair in Chinesehamster cells 1 74 Resistant cells yes AB000449 vaccinia related kinase1 76 275 75 Resistant cells yes U34994 protein kinase, DNA-activated, 63263 catalytic polypeptide 76 Resistant cells yes AA595596ADP-ribosyltransferase (NAD+; 104 N/A poly(ADP-ribose) polymerase)-like2 77 Resistant cells yes AB009010 ubiquitin C 147 340 78 Resistant cellsyes Y18483 solute carrier family 7 (cationic 61 261 amino acidtransporter, y+ system), member 8 79 Resistant cells yes U84570chromosome 21 open reading frame 2 91 290 80 Resistant cells yesAF007156 KIAA0751 gene product 82 281 81 Resistant cells yes AB007870KIAA0410 gene product 157 349 82 Resistant cells U80040 aconitase 2,mitochondrial 184 375 83 Resistant cells M64174 Janus kinase 1 (aprotein tyrosine 185 376 kinase) 84 Resistant cells yes AB011535 FATtumor suppressor (Drosophila) 65 265 homolog 2 85 Resistant cells yesAC005329 NADH dehydrogenase (ubiquinone) 71 270 Fe-S protein 7 (20 kD)(NADH- coenzyme Q reductase) 86 Resistant cells yes AF038172hypothetical protein FLJ11149 117 N/A 87 Resistant cells yes U68111protein phosphatase 1, regulatory 96 295 (inhibitor) subunit 2 88Resistant cells yes U71364 serine (or cysteine) proteinase 84 283inhibitor, clade B (ovalbumin), member 9 89 Resistant cells yes D89377msh (Drosophila) homeo box 94 293 homolog 2 90 Resistant cells Y00971phosphoribosyl pyrophosphate 186 377 synthetase 2 91 Resistant cellsAL050065 DNA DKFZp566M043 (from clone 187 N/A DKFZp566M043) 92 Resistantcells AF029670 RAD51 (S. cerevisine) homolog C 188 378 93 Resistantcells yes M31682 inhibin, beta B (activin AB beta 87 286 polypeptide) 94Resistant cells X63629 cadherin 3, type 1, P-cadherin 189 379(placental) 95 Resistant cells AB028957 KIAA1034 protein 190 380 96Resistant cells yes D87119 GS3955 protein 77 276 97 Resistant cells yesM14333 Homo sapiens cDNA FLJ32137 fis, 123 318 clone PEBLM2000479,highly similar to PROTO-ONCOGENE TYROSINE-PROTEIN KINASE FYN (EC2.7.1.II2) 98 Resistant cells yes L07540 replication factor C(activator 1) 5 97 296 (36.5 kD) 99 Resistant cells X74837 mannosidase,alpha, class 1A, 191 381 member 1 100 Resistant cells yes X90976runt-related transcription factor 1 93 292 (acute myeloid leukemia 1;aml1 oncogene) 101 Resistant cells AB018273 collagen, type I, alpha 2192 382 102 Resistant cells yes AF003837 jagged 1 (Alagille syndrome)105 303 103 Resistant cells yes AL050290 spermidine/spermine N1- 102 301acetyltransferase 104 Resistant cells yes AB014566 KIAA0666 protein 83282 105 Resistant cells Y15227 deleted in lymphocytic leukemia, 1 193383 106 Resistant cells yes X79067 zinc finger protein 36, C3H type- 64264 like 1 107 Resistant cells yes AF031824 cystatin F (leukocystatin)88 287 108 Resistant cells Y12661 VGF nerve growth factor inducible 194384 109 Resistant cells yes X74331 primase, polypeptide 2A (58 kD) 121316 110 Resistant cells yes AL043470 hypothetical protein FLJ10335 150N/A 111 Resistant cells yes U67733 phosphodiesterase 2A, cGMP- 151 343stimulated 112 Resistant cells AL049365 Homo sapiens mRNA; cDNA 195 N/ADKFZp586A0618 (from clone DKFZp586A0618) 113 Resistant cells yes L43821enhancer of filamentation 1 (cas- 81 280 like docking; Crk-associatedsubstrate related) 114 Resistant cells Y11395 LanC (bacterial1antibiotic 196 385 synthetase component C)-like 1 115 Resistant cellsyes L34059 cadherin 4, type 1, R-cadherin 100 299 (retinal) 116Resistant cells NM_004713 serologically defined colon cancer 197 386antigen 1 117 Resistant cells yes AB006626 histone deacetylase 4 86 285118 Resistant cells X76029 neuromedin U 198 387 119 Resistant cells yesU81561 protein tyrosine phosphastase, 74 273 receptor type, Npolypeptide 2 120 Resistant cells yes M87339 replication factor C(activator 1) 4 106 304 (37 kD) 121 Resistant cells yes U57352amiloride-sensitive cation channel 62 262 1, neuronal (degenerin) 122Resistant cells yes AF070530 hypothetical protein, clone 24751 80 279123 Resistant cells yes U19718 microfibrillar-associated protein 2 114310 124 Resistant cells yes U65676 Hermansky-Pudlak syndrome 119 314 125Resistant cells yes U52840 sema domain, seven 115 311 thrombospondinrepeats (type 1 and type 1-like), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 5A 126 Resistant cells AF045583 rubbylike protein 3 199 388 127 Resistant cells X85545 protein kinase,X-linked 200 389 128 Resistant cells yes AB014557 KIAA0657 protein 116312 129 Resistant cells yes M34182 protein kinase, cAMP-dependent, 99298 catalytic, gamma 130 Resistant cells X12534 RAP2A, member of RASoncogene 201 390 family 131 Resistant cells yes AB020661 KIAA0854protein 153 345 132 Resistant cells yes AI761647 Homo sapiens cloneIMAGE 21721 109 N/A 133 Resistant cells yes X80507 Yes-associatedprotein 1, 65 kDa 110 306 134 Resistant cells yes U14971 ribosomalprotein S9 120 315 135 Resistant cells yes X78817 Rho GTPase activatingprotein 4 79 278 136 Resistant cells yes D16815 nuclear receptorsubfamily 1, group 122 317 D, member 2 137 Resistant cells yes AB018306KIAA0763 gene product 108 305

TABLE 6 Highly SEQ ID Expressed Cells SEQ ID NO: of (Sensitive orGenbank NO: of Amino Gene # Resistent Accession # UniGene Title DNA Acid1 Sensitive cells AB014558 cryptochrome 2 (photolyase- 1 202 like) 2Sensitive cells NM_006979 HLA class II region expressed 2 203 gene KE4 3Sensitive cells M22489 bone morphogenetic protein 2 3 204 4 Sensitivecells AB023194 KIAA0977 protein 4 205 5 Sensitive cells U03688cytochrome P450, subfamily I 5 206 (dioxin-inducible), polypeptide 1(glaucoma 3, primary infantile) 6 Sensitive cells M88458 KDEL(Lys-Asp-Glu-Leu) 6 207 endoplasmic reticulum protein retention receptor2 7 Sensitive cells L13463 regulator of G-protein signalling 7 208 2, 24kD 8 Sensitive cells AF000560 Homo sapiens TTF-I interacting 9 210peptide 20 mRNA, partial cds 9 Sensitive cells L40802 hydroxysteroid(17-beta) 12 213 dehydrogenase 2 10 Sensitive cells X13916 low densitylipoprotein-related 13 214 protein 1 (alpha-2- macroglobulin receptor)11 Sensitive cells AF009674 axin 14 215 12 Sensitive cells M73077glucocorticoid receptor DNA 15 216 binding factor 1 13 Sensitive cellsU15655 Ets2 repressor factor 16 217 14 Sensitive cells M58603 nuclearfactor of kappa light 18 219 polypeptide gene enhancer in B- cells 1(p105) 15 Sensitive cells AI659108 Homo sapiens, clone 20 N/A IMAGE:3908182, mRNA, partial cds 16 Sensitive cells U72649 BTG family, member2 21 221 17 Sensitive cells X77909 nuclear factor of kappa light 23 223polypeptide gene enhancer in B- cells inhibitor-like 1 18 Sensitivecells AB006622 KIAA0284 protein 26 226 19 Sensitive cells AB029027KIAA1104 protein 27 227 20 Sensitive cells U51903 IQ motif containingGTPase 28 228 activating protein 2 21 Sensitive cells AB026891 solutecarrier family 7, (cationic 30 230 amino acid transporter, y+ system)member 11 22 Sensitive cells X60708 dipeptidylpeptidase IV (CD26, 34 234adenosine deaminase complexing protein 2) 23 Sensitive cells K03498 Homosapiens endogenous 41 241 retrovirus HERV-K104 long terminal repeat,complete sequence, and Gag protein (gag) and envelope protein (env)polynucleotides and polypeptides, complete cds 24 Sensitive cells D13413heterogeneous nuclear 47 247 ribonucleoprotein U (scaffold attachmentfactor A) 25 Sensitive cells M69023 tetraspan 3 48 248 26 Sensitivecells AF155654 Human putative ribosomal 51 251 protein S1 mRNA 27Sensitive cells L07261 adducin 1 (alpha) 54 254 28 Sensitive cellsL37033 FK506-binding protein 8 (38 kD) 56 256 29 Sensitive cells Y09846SHC (Src homology 2 domain- 57 257 containing) transforming protein 1 30Sensitive cells AF093420 hsp70-interacting protein 58 258 31 Sensitivecells U19775 mitogen-activated protein kinase 59 259 14 32 Resistantcells Y18483 solute carrier family 7 (cationic 61 261 amino acidtransporter, y+ system) member 8 33 Resistant cells U57352amiloride-sensitive cation 62 262 channel 1, neuronal (degenerin) 34Resistant cells U34994 protein kinase, DNA-activated, 63 263 catalyticpolypeptide 35 Resistant cells X79067 zinc finger protein 36, C3H 64 264type-like 1 36 Resistant cells AB011535 FAT tumor suppressor 65 265(Drosophila) homolog 2 37 Resistant cells U90902 Human clone 23612 mRNA66 N/A sequence 38 Resistant cells S37730 insulin-like growth factor 69268 binding protein 2 (36 kD) 39 Resistant cells AC005329 NADHdehydrogenase 71 270 (ubiquinone) Fe-S protein 7 (20 kD) (NADH-coenzymeQ reductase) 40 Resistant cells U81561 protein tyrosine phosphatase, 74273 receptor type, N polypeptide 2 41 Resistant cells AB000449 vacciniarelated kinase 1 76 275 42 Resistant cells D87119 GS3955 protein 77 27643 Resistant cells X06745 polymerase (DNA directed), 78 277 alpha 44Resistant cells X78817 Rho GTPase activating protein 4 79 278 45Resistant cells AF070530 hypothetical protein, clone 80 279 24751 46Resistant cells L43821 enhancer of filamentation 1 (cas- 81 280 likedocking; Crk-associated substrate related) 47 Resistant cells AF007156KIAA0751 gene product 82 281 48 Resistant cells AB014566 KIAA0666protein 83 282 49 Resistant cells AB006626 histone deacetylase 4 86 28550 Resistant cells M31682 inhibin, beta B (activin AB beta 87 286polypeptide) 51 Resistant cells AF031824 cystatin F (leukocystatin) 88287 52 Resistant cells U84570 chromosome 21 open reading 91 290 frame 253 Resistant cells X90976 runt-related transcription factor 93 292 1(acute myeloid leukemia 1; aml1 oncogene) 54 Resistant cells D89377 msh(Drosophila) homeo box 94 293 homolog 2 55 Resistant cells L07540replication factor C (activator 1) 97 296 5 (36.5 kD) 56 Resistant cellsM34182 protein kinase, cAMP- 99 298 dependent, catalytic, gamma 57Resistant cells L34059 cadherin 4, type 1, R-cadherin 100 299 (retinal)58 Resistant cells AL050290 spermidine/spermine N1- 102 301acetyltransferase 59 Resistant cells AA595596 ADP-ribosyltransferase(NAD+; 104 N/A poly(ADP-ribose) polymerase)- like 2 60 Resistant cellsAF003837 jagged 1 (Alagille syndrome) 105 303 61 Resistant cells M87339replication factor C (activator 1) 106 304 4 (37 kD) 62 Resistant cellsAB018306 KIAA0763 gene product 108 305 63 Resistant cells AI761647 Homosapiens clone IMAGE 109 N/A 21721 64 Resistant cells U19718microfibrillar-associated protein 2 114 310 65 Resistant cells U52840sema domain, seven 115 311 thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5A 66 Resistant cells AB014557 KIAA0657 protein 116 312 67Resistant cells AF038172 hypothetical protein FLJ11149 117 N/A 68Resistant cells U65676 Hermansky-Pudlak syndrome 118 313 69 Resistantcells U14971 ribosomal protein S9 119 314 70 Resistant cells X74331primase, polypeptide 2A (58 kD) 120 315 71 Resistant cells D16815nuclear receptor subfamily 1, 121 316 group D, member 2 72 Resistantcells M14333 Homo sapiens cDNA FLJ32137 122 317 fis, clone PEBLM2000479,highly similar to PROTO- ONCOGENE TYROSINE- PROTEIN KINASE FYN (EC2.7.1.112) 73 Resistant cells U68111 protein phosphatase 1, 96 295regulatory (inhibitor) subunit 2

TABLE 7 True Class for Error for Error Predicted Confidence BMS-A andBMS-A and True Class for True Class Error for Cell lines Class ScoreBMS-D BMS-D for BMS-B BMS-B for BMS-C BMS-C WiDr S 0.174 S S S SW1417 S0.264 S S S SW403 S 0.265 R * S S Caco-2 S 0.585 S S S SW837 R 0.515 R RS * HT29 S 0.827 S S S T84 R 0.025 R S * S * CCD-33Co S 1.000 S S S LOVOS 1.000 S S S CCD-18Co S 0.572 S S S LS174T S 0.905 S S S HCT15 R 0.419S * S * R CX-1 S 0.416 R * R * S Colo-205 R 0.756 R R R RKO RM13 R 0.696R R R DLD-1 R 0.593 R R R Colo-201 R 1.000 R R R HCT-8 R 0.760 R R RSK-Co-1 R 0.898 R R R MIP R 0.722 R R R Colo 320hfr R 1.000 R R R LS1034R 0.917 R R R Colo320DM R 0.950 R R R HCT116 R 0.010 R R R HCT116S542 R1.000 R R R LS180 R 0.589 R R R LS 513 R 0.795 R R R SW1116 R 0.695 R RR SW 948 R 0.523 R R R SW 480 R 1.000 R R R SW620 R 0.847 R R R

TABLE 8 True Class for Error for Error Predicted Confidence BMS-A andBMS-A and True Class Error for True Class for Cell lines Class ScoreBMS-D BMS-D for BMS-B BMS-B for BMS-C BMS-C WiDr S 0.146 S S S SW1417 S0.522 S S S SW403 S 0.506 R * S S Caco-2 S 0.679 S S S SW837 R 0.260 R RS * HT29 S 0.920 S S S T84 S 0.230 R * S S CCD-33Co S 0.979 S S S LOVO S0.969 S S S CCD-18Co S 0.488 S S S LS174T S 0.619 S S S HCT15 R 0.088S * S * R CX-1 S 0.522 R * R * S Colo-205 R 0.950 R R R RKO RM13 R 0.409R R R DLD-1 R 0.755 R R R Colo-201 R 0.870 R R R HCT-8 R 0.823 R R RSK-Co-1 R 0.817 R R R MIP R 0.781 R R R Colo 320hfr R 0.530 R R R LS1034R 0.815 R R R Colo320DM R 0.675 R R R HCT116 R 0.261 R R R HCT116S542 R0.782 R R R LS180 R 0.449 R R R LS 513 R 0.615 R R R SW1116 R 0.677 R RR SW 948 R 0.500 R R R SW 480 R 1.000 R R R SW620 R 0.795 R R R

TABLE 9 True Class for Error for Error Predicted Confidence BMS-A andBMS-A and True Class for Error for True Class for Cell lines Class ScoreBMS-D BMS-D BMS-B BMS-B for BMS-C BMS-C WiDr S 0.020 S S S SW1417 S0.286 S S S SW403 S 0.288 R * S S Caco-2 S 0.847 S S S SW837 S 0.178 R *R * S HT29 S 0.876 S S S T84 S 0.344 R * S S CCD-33Co S 0.870 S S S LOVOS 0.908 S S S CCD-18Co S 0.333 S S S LS174T S 0.468 S S S HCT15 R 0.426S * S * R CX-1 S 0.662 R * R * S Colo-205 R 0.498 R R R RKO RM13 R 0.402R R R DLD-1 R 0.834 R R R Colo-201 R 0.695 R R R HCT-8 R 0.300 R R RSK-Co-1 R 0.525 R R R MIP R 0.878 R R R Colo 320hfr R 0.474 R R R LS1034R 0.837 R R R Colo320DM R 0.436 R R R HCT116 R 0.433 R R R HCT116S542 R0.914 R R R LS180 R 0.562 R R R LS 513 R 0.726 R R R SW1116 R 0.589 R RR SW 948 R 0.298 R R R SW 480 R 0.861 R R R SW620 R 0.515 R R R

TABLE 10 Highly Accession # Unigene Title Expressed in AB014558cryptochrome 2 (photolyase-like) Sensitive cells AL031228 ring fingerprotein 1 Sensitive cells M22489 bone morphogenetic protein 2 Sensitivecells AB023194 KIAA0977 protein Sensitive cells U03688 cytochrome P450,subfamily I Sensitive cells (dioxin-inducible), polypeptide 1 (glaucoma3, primary infantile) AB026891 solute carrier family 7, Sensitive cells(cationic amino acid transporter, y+ system) member 11 X60708dipeptidylpeptidase IV (CD26, adenosine Sensitive cells deaminasecomplexing protein 2) K03498 Human endogenous retrovirus HERV-K22Sensitive cells pol and envelope ORF region D13413 heterogeneous nuclearribonucleoprotein Sensitive cells U (scaffold attachment factor A)M69023 tetraspan 3 Sensitive cells

TABLE 11 Highly Accession # Unigene Title Expressed in AB014558cryptochrome 2 (photolyase-like) Sensitive cells NM_006979 HLA class IIregion expressed gene KE4 Sensitive cells M22489 bone morphogeneticprotein 2 Sensitive cells AF009674 axin Sensitive cells AB006622KIAA0284 protein Sensitive cells AB023194 KIAA0977 protein Sensitivecells U03688 “cytochrome P450, subfamily I Sensitive cells(dioxin-inducible), polypeptide 1 (glaucoma 3, primary infantile)”L40802 hydroxysteroid (17-beta) dehydrogenase 2 Sensitive cells Y18483“solute carrier family 7 (cationic amino Resistant cells acidtransporter, y+ system), member 8” U90902 Human clone 23612 mRNAsequence Resistant cells S37730 insulin-like growth factor bindingResistant cells protein 2 (36 kD) X79067 “zinc finger protein 36, C3Htype-like 1” Resistant cells D87119 GS3955 protein Resistant cellsM31682 “inhibin, beta B (activin AB beta Resistant cells polypeptide)”AC005329 NADH dehydrogenase (ubiquinone) Resistant cells Fe—S protein 7(20 kD) (NADH- coenzyme Q reductase)

TABLE 12 Highly Accession # Unigene Title Expressed in AB014558cryptochrome 2 (photolyase-like) Sensitive cells M22489 bonemorphogenetic protein 2 Sensitive cells AF009674 axin Sensitive cellsAB006622 KIAA0284 protein Sensitive cells AB023194 KIAA0977 proteinSensitive cells U03688 “cytochrome P450, subfamily I (dioxin- Sensitivecells inducible), polypeptide 1 (glaucoma 3, primary infantile)” L40802hydroxysteroid (17-beta) dehydrogenase 2 Sensitive cells X77909 nuclearfactor of kappa light polypeptide Sensitive cells gene enhancer inB-cells inhibitor-like 1 U19775 mitogen-activated protein kinase 14Sensitive cells U51903 IQ motif containing GTPase activating Sensitivecells protein 2 X60708 “dipeptidylpeptidase IV (CD26, Sensitive cellsadenosine deaminase complexing protein 2)” M69023 tetraspan 3 Sensitivecells AF155654 Human putative ribosomal protein Sensitive cells S1 mRNAY18483 “solute carrier family 7 (cationic amino Resistant cells acidtransporter, y+ system), member 8” U34994 “protein kinase,DNA-activated, Resistant cells catalytic polypeptide” U90902 Human clone23612 mRNA sequence Resistant cells S37730 insulin-like growth factorbinding Resistant cells protein 2 (36 kD) X79067 “zinc finger protein36, C3H Resistant cells type-like 1” M31682 “inhibin, beta B (activin ABbeta Resistant cells polypeptide)” AC005329 NADH dehydrogenase(ubiquinone) Resistant cells Fe—S protein 7 (20 kD) (NADH- coenzyme Qreductase) M34182 “protein kinase, cAMP-dependent, Resistant cellscatalytic, gamma” AF007156 KIAA0751 gene product Resistant cells M14333“Homo sapiens cDNA FLJ32137 fis, Resistant cells clone PEBLM2000479,highly similar to PROTO-ONCOGENE TYROSINE- PROTEIN KINASE FYN (EC2.7.1.112)” X06745 “polymerase (DNA directed), alpha” Resistant cellsU84570 chromosome 21 open reading frame 2 Resistant cells

TABLE 13 SEQ ID SEQ ID NO: NO: DNA Forward Forward Seq # Accession #Forward Primer Primer Reverse Primer Primer 1 AB014558CTGAACCCTTTGGGAAAGAAC 391 AAGCGCTTGTATGTAAGGGGT 592 2 NM_006979AGGCTTAGACCTGCGTGTGT 392 CTGTCCACTGCTCCTCCTTC 593 3 M22489GCAGTTTCCATCACCGAATTA 393 ATCAAAACTTTCCCACCTGCT 594 4 AB023194AATCCACTGCTTTCATCATGG 394 TGTTCAGCTGACAACAGATCG 595 5 U03688AACTGTCCATCAGGTGAGGTG 395 TTCATTGGGCCCTTTAAGTCT 596 6 M88458CTTTGAGGGCTTCTTTGACCT 396 TTATGCTGGCAAACTGAGCTT 597 7 L13463GCCCAGAAAAGGGTATACAGC 397 CTGGGCTCCCTTTTACATTTC 598 8 U21551GTGTACCGGAGAAGGAGGATC 398 TTATTGGGGTCTGGTTTTTCC 599 9 AF000560TATTAGGGCCCGTTCACTTCT 399 CCTCTGCAGTTCTCTCCATTG 600 10 AF102265TGATCATGTCATGTTTCGCAT 400 CATTTGCTAAACAGGTGGCAT 601 11 X06272ATGGCACCTCTCCCTAGGATA 401 CTGATGCTTTGGGGTAAACAA 602 12 L40802ACAAGTGGCATTGGACTCATC 402 CAGTTTCCCAGTTTCCCTTTC 603 13 X13916GATTGCCTGGACAACAGTGAT 403 ACAAGTGGCATTGGACTCATC 604 14 AF009674AAGAGCTTCATAAAGGGCTGC 404 TGGTCACTACAGACTTTGGGG 605 15 M73077GTCGTGACTCCAGAGAAGCC 405 AGGTCCAGGTTGTGGTCTTG 606 16 U15655TGTAGTGATGGCACGTCAGAG 406 GGGATAGACTCGGAAGACACC 607 17 AB014520AAGGCCATTCTGAGTATCCGT 407 TGGTCTTCCAGATGTGTAGGG 608 18 M58603CAGGTCCAGGGTATAGCTTCC 408 TTTGTCACAACCTTCAGGGTC 609 19 X76104AAGAACCGAGAAGGAGAGACG 409 TTACCTCCATCTGACACCGTC 610 20 AI659108CACTGTCACAACCCCAATTTC 410 ACCACTGTACGGAATGTGAGG 611 21 U72649AGAGTGAAAAGGCCTCTCCTG 411 CCTTCCATCCTAACCCCAATA 612 22 M64571TACGGTATGTCTCCCTGCAAC 412 CCTTGGCTAGCTCTAAGGGAA 613 23 X77909GTGGGAGCGAAAGTTGTAACA 413 TTTGAGATGTGGAACCAGGTC 614 24 M34064GGGTAATCCTCCCAAATCAAA 414 TCCATACCACAAACATCAGCA 615 25 AL050345GGTTGGCAATAGAAGGTGACA 415 GAGCACCAAAAAGCTCATCAG 616 26 AB006622TACACCTCCACCACTCAGACC 416 GAAACCATAAGGGTCAGGCTC 617 27 AB029027AAGTATCACGAGAAGCAGGCA 417 CAGACAAGAGGCATCTTGAGG 618 28 U51903GCTGCAGTGGACCATATCAAT 418 CACAGCAATCAAAAGCTCCTC 619 29 AF041259AAAAACAAACCGATGTTGCTG 419 GGCATCTCCTTAAGCTGCTTT 620 30 AB026891ACCTTCCAGAAATCCTCTCCA 420 ACCTGGCAAAACTGAGGAAAT 621 31 AB007960GGGGCCATTGCTATAAATCAT 421 GGGCAAGAACTGTGTGCATAT 622 32 D63390TGAAACCAGAAGGGGACTTTT 422 CATAATCCACAGAAAGGCCAA 623 33 L10678TACCAACGGTTTGACTCTTGG 423 ACCTTGACTCTTTGTCCGGAT 624 34 X60708AAAAACACAGCAAGGGTGATG 424 TCTTTTAACAGGGCAAGCTGA 625 35 Y15521AATACTCTCTGCACGGCTTCA 425 GGTAGTACGCATCCTGGAACA 626 36 AI038821ATCCAATAATTGGGTGGGATC 426 AGGCTGTGCACAGACTGTCTT 627 37 X84740CAACACGAAGACCCAGATCAT 427 AAATGCGACTGAAAAGCTTCA 628 38 M23115AACATGAAACAGTTCATCCGC 428 GCAGCTGAACAGGAATCAAAG 629 39 U79287GCTCATGCTCCTGTACTCGTC 429 CCATGCCAGAAACTTGTTGTT 630 40 Y12781CTCTTCCTTCTGTTGCTCCCT 430 CGTGGGAGATTGTGTCTTCAT 631 41 K03498ACAGATGAAGTTGCCATCCAC 431 AGCTGCAAGCAGCATACTCTC 632 42 AF030335TACTGGTGGTTGAGTTCCTGG 432 CAGCTGGACAGAGAAGACCAC 633 43 X93209GATGCAATTGACCGTGAAGTT 433 TCTAGCAAGGCTTCCAAACAA 634 44 AF068744GACAGCGAAGGAGACTCGTTT 434 TGAAACAGAATCTGGACCCTG 635 45 AF072247TCTGTCCCAGCTCCTTGAGACT 435 GACGTGCCTCGACTGTGTTA 636 46 U41344TTCCACCCAGTTGAAAGACAC 436 GAGAGAAGGGGACACCAAGTC 637 47 D13413GGAAGAGGAGGAGGAAGGAAT 437 ATCTTCCCCTTCCTGGAAAC 638 48 M69023CTGGCATTTGAGCTATTCAGC 438 TCAACTATGCATAGGTTCCGC 639 49 J04599CTGAAGTCTGTGCCCAAAGAG 439 ATCTTGTTGTTCACCAGGACG 640 50 U79267GTCAGCGAGATGGTGAAGAAG 440 CAGACAAAGACAAAGGCTTGC 641 51 AF155654AGAAGGCTGAGGAGAAAGCC 441 CTGTGAACCACCGATCTCCT 642 52 X12794CAGGACTCTGGCTTCTCTCCT 442 CTGTCCTAGGATTGGACCCTC 643 53 U51166TTTCAGTGGCATTCCTAATGG 443 CTGCAGCATTTAAGCAGAGCT 644 54 L07261GTGACTGCATCCAGTTTGGTT 444 TGCAGCATAAATTGCAGAGTG 645 55 U97188AAACCATGTGATTTGCCTCTG 445 GCATTTTCTTTACGGTGGACA 646 56 L37033ATCCTCAACCCCATCCAGTAC 446 CAGCTCACTCAGGTCATCCTC 647 57 Y09846ACCTCATCAGCTACCACATGG 447 AGGGCATCTTCTGGAAGAGAG 648 58 AF093420ATCTCCTGTCTGGTCCGAGAG 448 TGCTGATTTGACCTTGAGCTT 649 59 U19775CGAGCTGTTGACTGGAAGAAC 449 TCGGCATCTGAGTCAAAGACT 650 60 J04027TAAAGCAGTTATGTGGGGACG 450 AGGGAAGCGAGTGTATCCATT 651 61 Y18483CATCAACTACGTGGGCTTCAT 451 CTCTGACCACAGGCTGAAGAC 652 62 U57352AGGATGAGTACCTGCCCATCT 452 TATGTGAGCCTCTGCTCCTGT 653 63 U34994CAGAAACGATCAACACGGAAT 453 GGTCTAACATGCCGTTCAAAA 654 64 X79067TTGCAAAGGCATCTTCTCAGT 454 CTGCCTTTGCTTTTTCTTGTG 655 65 AB011535TCTAGAGCAGTGACCCTGGAA 455 AGTTGAGCCAGCACAGTCAAT 656 66 U90902ACTGTACCCTTCCCTCTTCCA 456 GACCAGCCATAGACCAAAACA 657 67 AB009282GAACTGTGGCTTGTGATCCAT 457 TCGGATGGATATCACCAATGT 658 68 AJ001685TCAGGCTTCCTAAAAGTTGCA 458 TTTCAACCTCCCTTAGGCATT 659 69 S37730TCCCTCGCACATTCAGATAAC 459 TAACCACAGCCCTACTCCCTT 660 70 U37518AGAGAAGGAAGGGCTTCAGTG 460 ATCTGCTTCAGCTCGTTGGTA 661 71 AC005329CTCCTTCTGCTGACATTGGAG 461 GCAATGTCTGAAAACACGGTT 662 72 AB009426TGTGGCTTGGAGATGAATAGG 462 TTTTGGGGTACCTTGTGAACA 663 73 X70340GAATGACTCAAATGCCCAAAA 463 AAGCCTGGTAAATCAATGGCT 664 74 U81561GTATGACCGAGGAGTCCCTTC 464 ATGTCGATCAGGACGTAGGTG 665 75 X70040CACACCCCTGCCTATTCTGTA 465 GTGGCACACAGGATTCATCTT 666 76 AB000449ATGGCCTTGCTTATCGGTACT 466 CCATTGGATCATGCAATAACC 667 77 D87119AAGGAACAGTTGGCCAAGAAT 467 GTCTGTGTGCACCGAATTTTT 668 78 X06745AATGCTACCTGTGGTCGAATG 468 TCTTTCCAGGTGTGTTCCAAC 669 79 X78817AACAAGACTCTGAAGGCGACA 469 GTCTGAGCTGGTGGACTTGAG 670 80 AF070530ACCACATGTGGAACCAGAGAG 470 TGACCTCATCTTCCACTGTCC 671 81 L43821GACAGGCCATGGCTACGTATA 471 ACTGAAAACACAGGGCCTTTT 672 82 AF007156CCCACCATAGAATTTCTGCAA 472 AGACTGAAGCCTGTCCTGTCA 673 83 AB014566AGGAGGAAGAAGAACGTCGAG 473 GTCAAACACTTCTCCTGAGCG 674 84 U71364ATTGTTGATGCCTTCCAACAG 474 CCTTCTTCATTCACCTCCACA 675 85 U93305TCTACCTGCCACCCCTACTTT 475 GCCTGTGCATCTATTCTCTGC 676 86 AB006626AGAACGGTCTTGGGACTTGTT 476 CAGAGGGCTATGCAGAGAATG 677 87 M31682GCAATGACCGTTTGACTGTTT 477 ATTTAGCCCCCTCTTCTCTCC 678 88 AF031824AAGACCACAGCCATGACAAAC 478 TGTTAGGAGGTGCTACCATGC 679 89 AF035299GAGGGCTCTACCTGAGAAGGA 479 GGATTGTCCTTCCCTTGACTC 680 90 X82207TAACCCTTTTTGGTCTTGGCT 480 CATGTCCTGTGTAGAGGGGAA 681 91 U84570GTTAGGTACTGGCTAACCGGG 481 AAATCCTCCCTTTAAGAGCCC 682 92 AA873266GGGAAAGTCCAGGTGGTAGAG 482 AAGATTGCCTTCTGCAAGTCA 683 93 X90976CCATGTCTGACCTGCAAAAAT 483 AGGCTGGTTTTGAGTTGGAAT 684 94 D89377CTCCAGCTTCAGTCTCCCTTT 484 GGTCTTCCTTAGGACAGGTGG 685 95 M57730CTGGAACAGTTCAAATCCCAA 485 CAGCTGGTACTCCTCATGCTC 686 96 U68111CAAGTGACCAACAGCAAAACA 486 TGTGAAGAACAAGAAGCAACG 687 97 L07540AGTCAGACATTGCCAACATCC 487 CTCAATGTCTGCCATTTTGGT 688 98 M65066AGAGTGGGTGACCAACATCAG 488 GAACTCCTCGTACATCTTGCG 689 99 M34182AAGCCCAGATATTTGGAGGAA 489 GTTTAAAACAGGCAGAAGGGG 690 100 L34059CCATGGAGGTCTTCAGCATTA 490 TGTCATTCATGTCGATGACGT 691 101 L25665ATCGATACCGACTGCATTTTG 491 TCGAGGAAAGTCCAGAACTGA 692 102 AL050290GGACTCCGGAAGGTTACAGTC 492 AACCAACAATGCTGTGTCCTC 693 103 X67325ACTCTCCGGATTGACCAAGTT 493 CTGGCATGGTTCTCTTCTCTG 694 104 AA595596TCACCACAGCTGAAGGAAATT 494 TGGGAGTACAGTGCCATTAGG 695 105 AF003837CCTGTAACATAGCCCGAAACA 495 AGTTGTCTCCATCCACACAGG 696 106 M87339TTCCCTGGGTGGAAAAATATC 496 CAGGTGGTCCGTAAAACAAGA 697 107 AI813532TCTGACATCTTGATTCCAGGG 497 GGCAGGGTGATAAATTGTTGA 698 108 AB018306AGCGGAAAATGAAGCTAGAGG 498 GATCCGTTCATAGATCCCCAT 699 109 AI761647GAAAAGACCCAAGGTTTCTGG 499 CCAAAGGCTGGTAGGAGATTC 700 110 X80507CAACTGCAGATGGAGAAGGAG 500 GACACTGGATTTTGAGTCCCA 701 111 Y16241TGGTTTGGCAACAGGTATCTC 501 GAAAGTCAGGTGCATGCTCTC 702 112 D67031TGAATATGGGTTCCCATCAAA 502 GTGCCTAGGCTTCTCTCGAAT 703 113 J05581CCAGTCTCCTTTCTTCCTGCT 503 CAGTAGAGCTGGGCACTGAAC 704 114 U19718CTCTTCCTGCTATTCCTGCCT 504 TAGTCTGGGTTGTCGATCTGG 705 115 U52840GTTCATTCTGGTGCATGAGGT 505 CTGCTTGAGGTCTGATTCAGG 706 116 AB014557GTTTGAGTGCAGGACAGAAGG 506 ACACTCGGAAGTTATGGCATG 707 117 AF038172TTACAGCTTGAGGGAAAAGCA 507 GGCCCAAAGCATCTGTAATCT 708 118 AB014529CTGAAGCCATTGAAAAAGCTG 508 CTAACAGCATGCCCAACATTT 709 119 U65676AGGCTTTCTCCAAAAGTGAGC 509 TCAGAAAGTTCAGCCGGTAGA 710 120 U14971TTGACTTCTCTCTGCGCTCTC 510 TGTTTATTTGGCAGGAAAACG 711 121 X74331CCAAACCAAGTGTCCAGAAAA 511 CAGGCTATTGAGGAAAAAGGG 712 122 D16815GTGAATGCAGGAGGTGTGATT 512 CCTTCAATATTGGCGAGATCA 713 123 M14333CCTCTGTGAAGCATTCGAGAC 513 GGATTGTTGGCACTGGAGTAA 714 124 M28668ATTATCACCAGCACCAGTTCG 514 GGGTTGACATAGGTGCTTGAA 715 125 W29065GAGAGAATAACCATCCGGGAC 515 AAGATGGGGAGATGTGGAAAC 716 126 AL050025CAGGTACGAATTTTGCGGTTA 516 TCGCAATCCACTCTCTGACTT 717 127 AF000561TACGAGTGCAACATCTGCAAG 517 TCTTCAGGTCGTAGTTGTGGG 718 128 AB008515ATGGGAATTGGTCTTTCTGCT 518 CACAACAGCCAATGACATCAC 719 129 AJ011736GAGGTCCTGGATAGCTCCAAC 519 AAGCTTCTGTCCCCTGAAGAG 720 130 J00277GCTGAAAGGAAAGCAGATGTG 520 GGACTTCCCAGTCTTGTCCTC 721 131 Y10055AGGCCTCATTGAGGTGGTACT 521 CTTGGACTTCAGCCAGTTGAG 722 132 W26652TTTTCCGTTGCAGCTGTTAAT 522 CTCACTTAGCGAAAGTGACGG 723 133 M11717GACGAGTTTGAGCACAAGAGG 523 AGGCCCCTAATCTACCTCCTC 724 134 L32976ACCCTGAAGATCACCGACTTT 524 GAAGGTGGAGGCCTTGATAAC 725 135 M29893CTAGATGGGGAGGAAGTCCAG 525 GAGAAAACACAGAGGAACCCC 726 136 S70154TGAGATGCCACTGACTGACAG 526 CTTGCCATTTTGTGGCTACAT 727 137 D83542ACTTCATCAATGATGGCTTGG 527 CCCCCAGTCTCTGAGGTAGTC 728 138 Z74615GTACATCAGCAAGAACCCCAA 528 TGGTAGGTGATGTTCTGGGAG 729 139 M96684GACTACGGAGTGGAGGAGGAG 529 GCATAAACACGCCGTACTTGT 730 140 D12763CCTGTGCCATAAAATGTGCTT 530 GGGAGGACGAACAATTTAAGC 731 141 S67070AGCCATAGTTGAGCCCTGATT 531 GATGCTGCTACCTCTGGAGTG 732 142 X66435CTCACCTCAGCATTTAGCAGG 532 GTGCCACACCAGTTCTTGAAT 733 143 U25138AAGTCATTGCCTGCTCAAGAA 533 CAGTTGAGTGGGGACAGGTAA 734 144 D85131ACTGGTGAGGTTTGTCCAATG 534 GGAGCTTGTACCAAGGGACTC 735 145 M36089CAGACAAAGATGAGGCAGAGG 535 CTGTGACTGGGGATGTCTTGT 736 146 AC004472TCTACTCTCAACTCCAGGGCA 536 TGGAGCTGACCTGACAGAGAT 737 147 AB009010GAATGTCAAGGCAAAGATCCA 537 TGATGGTCTTACCAGTCAGGG 738 148 D26158CGCTTTGACAAGAGGATTGAG 538 TGACTGGTAGAGGTGGGTGAG 739 149 AL035307CTTTGTCATCATTGCCCAACT 539 TGGTTCATTTTGGCTCTCATC 740 150 AL043470TACACTTCCACAGTCAGCACG 540 AAGGACAGGTATGATGATGCG 741 151 U67733CAAGTACTACCTTCCTGGGCC 541 TGCTCAGACAAAGAATGGCTT 742 152 D28118TCACCATCACATCTCCAATGA 542 TTTACCAAGGCGGTGATGTAG 743 153 AB020661CATTAGGACACGTCATGCCTT 543 GAGTTGATCATCGTGGCATTT 744 154 W72186TATAGCAACAGCGTGTGCAAG 544 TTTCATGCACACACACACATG 745 155 U97067ATGAGCACAGAGAACGCATCT 545 GTTCTTCAGCGATGCTTTTTG 746 156 AF061261CCCAAAAGTTGTCAGGTTGAA 546 TTTTAAGTGTGTCGGAGGGTG 747 157 AB007870ACATGAATATGGGCACAGAGC 547 TCTGCAGCCTTCAGAACAAAT 748 158 AB014519CTTAGCAATGAGATGCAAGCC 548 GCATTTTTGTGCTGAAGAAGC 749 159 AB011123CATTGAGTTGAGCTGGCCTAG 549 GCTTGTTTCCCAGAAGCTCTT 750 160 U09578TTTCATGGCTGATCAGAGCTT 550 TAAAACCCAGCAGTGTTGTCC 751 161 Y17711TCTGTGTTAGTCCCTGGGATG 551 GCTCACTAAGCACAGGGTCAC 752 162 L13972TCCTCTCGGTCATCTTCTCAA 552 TGGGTTGTTCTCCCAGTAGTG 753 163 AF055009CAAGTACCTGAGTGAGGCCTG 553 AGCAAGAACTCCTCTTCTGGG 754 164 AF007155TTCCAGAACTTCTCCCTCCAT 554 GAGGCACTCAGTCTCCCTTCT 755 165 AB018324AGCACCTTATCCGAAGGATGT 555 AGTCGCAGAACCTGCTCATTA 756 166 AB023154ATGAGGTCCACCACAAGACAG 556 AAAGCTTTTCTGGCCTCAGTC 757 167 X78992ATCCAGAAACATGTCGACCAC 557 GCATGTTGTTCAGGTTGAGGT 758 168 M60299CTTGCTCCTTTCTTTTGCATG 558 ACTCCACTGAACCCCTCTGTT 759 169 M80482TAAAAAGTGCGTGGATGAACC 559 GTGAATGCACTCTTCTCTGCC 760 170 U47025GGTCAGTGACGAGGTGTTCAT 560 GGATCCTCTTCACATGCACAT 761 171 Y10032AGGATGGGTCTGAACGACTTT 561 AAGGGTTGGCATTCATAAGCT 762 172 U57057GGGAACTGTTTAAGGGTGAGC 562 CACGGAGTCGTAGCAGTTCTC 763 173 X90392CTCATTTGTCCCAACAGCATT 563 TGAGTTAGGGGTGTTTGATGC 764 174 AA418437GAGACAGGGCTGTGTTCTGAG 564 AGCAGTAGCAGTGAGAGCGAG 765 175 X84373TCCTCCTTGCGTAGAAGTTGA 565 ATGGTTTTCTGTGGTGAGTGC 766 176 XM_212189TGTTCACTTTTGTTGCGGAG 566 CTGCATGGAGAAGATGACGA 767 177 AF109134GGGAGGTCGAGGTGTTTAAAA 567 GGAAGCGCTTCTGGTAGTTCT 768 178 M64788ATTTGGGTTCTGCTGTGTGAC 568 AAGGCAGGTGCATGTAATCAC 769 179 U43368AAGAGGGGTCACATACCAGCT 569 GGGGTCACAGTTCTTGTACCA 770 180 X13293ACCAGTCTGTCCTTCCTGGAT 570 GCTCCAATGTGTCCTGTTTGT 771 181 AB014511CTCAGTATCGTCTCCAGCCAG 571 ATAAAGCGGGATCACACCTCT 772 182 AB014585CCGTGAGGAGAGGACAGAAG 572 CACTTCTGGAACAGGTGGGT 773 183 AB011114CGATTCCATCATGTCACTGTG 573 CAGCAGCATGTAGTGTTTCCA 774 184 U80040CACTTCCGTGTTCCCTTACAA 574 TAGTTGGTCATAATGGCAGCC 775 185 M64174TGAAGCCTGAGAGTGGAGGTA 575 CTCCGTCTTCTGTGCAGATTC 776 186 Y00971ACTTGTGGCCAATATGCTGTC 576 GCTCCGCATACAAATTATCCA 777 187 AL050065CCTTTCATGTCTCCCACTGAA 577 GCCCACAGATACTTGACCGTA 778 188 AF029670TCCGAGCTTAGCAAAGAAGTG 578 CCTGCTCAAGAAGTTCCAGTG 779 189 X63629TGTTGAATAAGCCACTGGACC 579 GTAAACTTGGGCTTGTGGTCA 780 190 AB028957TGTTGTGACGTTGAAAATCCA 580 AGGGCATTCTTTGGCTAATGT 781 191 X74837TTTCGAAAGAAAGCAGTGGAA 581 GGGTTTCCTGATAAGTGGCTC 782 192 AB018273ACGACGTTGGATGAAGAAATG 582 CCTCCTCAGGCTTCAGAAGTT 783 193 Y15227AAATACGGGTCCTGCTTAGGA 583 GAGGCTTAAGTGCGATAACCC 784 194 Y12661TCTATTTTTCAGTCTCCGGCA 584 TACCGGCTCTTTATGCTCAGA 785 195 AL049365TTCTTGGTCTTGGAACTCCCT 585 TTAAAGTGCCAGTATCGGTGG 786 196 Y11395CAGGATCAGCTTCCTCCTTCT 586 TTCACAAGAAAGGCAGAGCAT 787 197 NM_004713GGTGACTCGAGCAGTGATGA 587 TTTGGGGTTGAAGGTGAGAC 788 198 X76029TGGAGGAGCTTTGCTTTATGA 588 CAACATTTGACTTGCCCAACT 789 199 AF045583TCTTCTTGCAGCTAGAAAGCG 589 CAGATGCCACGGTCATAAACT 790 200 X85545ACCTTTTCTTCACGTGGAGGT 590 TGAGAAGCACAGGCTGAAAAT 791 201 X12534CAGAGCTTCCAGGACATCAAG 591 GAAGTTTCCATAAAGGGGCAG 792

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals,abstracts and internet websites cited herein are hereby incorporated byreference in their entirety to more fully describe the state of the artto which the invention pertains.

As various changes can be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

1. A predictor set comprising a plurality of polynucleotides whoseexpression pattern is predictive of the response of cells to treatmentwith a compound that modulates protein tyrosine kinase activity ormembers of the protein tyrosine kinase pathway.
 2. The predictor setaccording to claim 1 wherein the polynucleotides are selected from thegroup consisting of: a.) the polynucleotides provided in Table 3; b.)the sensitive predictor polynucleotides provided in Table 3; c.) theresistant predictor polynucleotides provided in Table 3; d.) thepolynucleotides provided in Table 4; e.) the sensitive predictorpolynucleotides provided in Table 4; f.) the resistant predictorpolynucleotides provided in Table 4; g.) the polynucleotides provided inTable 5; h.) the sensitive predictor polynucleotides provided in Table5; i.) the resistant predictor polynucleotides provided in Table 5; j.)the polynucleotides provided in Table 6; k.) the sensitive predictorpolynucleotides provided in Table 6; and l.) the resistant predictorpolynucleotides provided in Table 6;
 3. The predictor set according toclaim 2 wherein the plurality of polynucleotides comprise the number ofpolynucleotides selected from the group consisting of: a) at least about5 polynucleotides; b.) at least about 10 polynucleotides; c.) at leastabout 15 polynucleotides; d.) at least about 20 polynucleotides; e.) atleast about 25 polynucleotides; and f.) at least about 30polynucleotides.
 4. The predictor set according to claims 3 wherein theplurality of polynucleotides comprise a member of the group consistingof: a) the polynucleotides provided in Table 10; b.) the sensitivepredictor polynucleotides provided in Table 10; c.) the resistantpredictor polynucleotides provided in Table 10; d.) the polynucleotidesprovided in Table 11; e.) the sensitive predictor polynucleotidesprovided in Table 11; f.) the resistant predictor polynucleotidesprovided in Table 11; g.) the polynucleotides provided in Table 12; h.)the sensitive predictor polynucleotides provided in Table 12; and i.)the resistant predictor polynucleotides provided in Table
 12. 5. Thepredictor set according to claim 4 wherein the protein tyrosine kinasesare selected from the group consisting of: Src, Fgr, Fyn, Yes, Blk, Hck,Lck and Lyn, Bcr-abl, Jak, PDGFR, c-kit and Ephr.
 6. The predictor setaccording to claim 5 wherein the compound is elected from the groupconsisting of: a) antisense reagents directed to said polynucleotides;b.) antibodies directed against polypeptides encoded by saidpolynucleotides; and c.) small molecule compounds.
 7. The predictor setaccording to claim 5 wherein the compound is selected from the groupconsisting of: a.) BMS-A; b.) BMS-B; c.) BMS-C; and d.) BMS-D.
 8. Thepredictor set according to claim 1 wherein said cells are colon cancercells.
 9. A predictor set comprising a plurality of polypeptides whoseexpression pattern is predictive of the response of cells to treatmentwith compounds that modulate protein tyrosine kinase activity or membersof the protein tyrosine kinase pathway.
 10. The predictor set accordingto claim 9 wherein the polypeptides are selected from the groupconsisting of: a.) the polypeptides provided in Table 3; b.) thesensitive predictor polypeptides provided in Table 3; c.) the resistantpredictor polypeptides provided in Table 3; d.) the polypeptidesprovided in Table 4; e.) the sensitive predictor polypeptides providedin Table 4; f.) the resistant predictor polypeptides provided in Table4; g.) the polypeptides provided in Table 5; h.) the sensitive predictorpolypeptides provided in Table 5; i.) the resistant predictorpolypeptides provided in Table 5; j.) the polypeptides provided in Table6; k.) the sensitive predictor polypeptides provided in Table 6; and l.)the resistant predictor polypeptides provided in Table
 6. 11. Thepredictor set according to claim 10 wherein the plurality ofpolypeptides comprise the number of polypeptides selected from the groupconsisting of: a.) at least about 5 polypeptides; b.) at least about 10polypeptides; c.) at least about 15 polypeptides; d.) at least about 20polypeptides; e.) at least about 25 polypeptides; and f.) at least about30 polypeptides.
 12. The predictor set according to claims 11 whereinthe plurality of polypeptides comprise a member of the group consistingof: a.) polypeptides provided in Table 10; b.) the sensitive predictorpolypeptides provided in Table 10; c.) the resistant predictorpolypeptides provided in Table 10; d.) the polypeptides provided inTable 11; e.) the sensitive predictor polypeptides provided in Table 11;f.) the resistant predictor polypeptides provided in Table 11; g.) thepolypeptides provided in Table 12; b.) the sensitive predictorpolypeptides provided in Table 12; and i.) the resistant predictorpolypeptides provided in Table
 12. 13. The predictor set according toclaim 12 wherein the protein tyrosine kinases are selected from thegroup consisting of: Src, Fgr, Fyn, Yes, Blk, Hck, Lck and Lyn, Bcr-abl,Jak, PDGFR, c-kit and Ephr.
 14. The predictor set according to claim 13wherein the compound is selected from the group consisting of: a.)antisense reagents directed to polynucleotides encoding saidpolypeptides; b.) antibodies directed against said polypeptides; and c.)small molecule compounds.
 15. The predictor set according to claim 13wherein the compound is elected from the group consisting of: a.) BMS-A;b.) BMS-B; c.) BMS-C; and d.) BMS-D.
 16. The predictor set according toclaim 9 wherein said cells are colon cancer cells.
 17. A method forpredicting whether a compound is capable of modulating the activity ofcells, comprising the steps of: a.) obtaining a sample of cells; b.)determining whether said cells express a plurality of markers; and c.)correlating the expression of said markers to the compounds ability tomodulate the activity of said cells.
 18. The method according to claim17 wherein the plurality of markers are polynucleotides.
 19. The methodaccording to claim 18 wherein the polynucleotides are selected from thegroup consisting of: a.) the polynucleotides of claim 1; b.) thepolynucleotides of claim 2; c.) the polynucleotides of claim 3; and d.)the polynucleotides of claim
 4. 20. The method according to claim 19wherein the compounds are a member of the group consisting of: a.) thecompounds according to claim 5; b.) the compounds according to claim 6;and c.) the compounds according to claim
 7. 21. The method according toclaim 18 wherein the cells are colon cancer cells.
 22. The methodaccording to claim 17 wherein the plurality of markers are polypeptides.23. The method according to claim 22 wherein the polypeptides areselected from the group consisting of: a.) the polypeptides of claim 9;b.) the polypeptides of claim 10; c.) the polypeptides of claim 11; andd.) the polypeptides of claim
 12. 24. The method according to claim 23wherein the compounds are a member of the group consisting of: d.) thecompounds according to claim 13; e.) the compounds according to claim14; and f.) the compounds according to claim
 15. 25. The methodaccording to claim 19 wherein the cells are colon cancer cells.
 26. Aplurality of cell lines for identifying polynucleotides and polypeptideswhose expression levels correlate with compound sensitivity orresistance of cells associated with a disease state.
 27. The pluralityof cell lines according to claim 26 wherein said cell lines are coloncancer cell lines.
 28. The plurality of cell lines according to claim 27wherein said cell lines comprise one or more cell lines provided inTable
 1. 29. A method of identifying polynucleotides and polypeptidesthat predict compound sensitivity or resistance of cells associated witha disease state, comprising the steps of: a.) subjecting the pluralityof cell lines according to claim 28 to one or more compounds; b.)analyzing the expression pattern of a microarray of polynucleotides orpolypeptides; and c.) selecting polynucleotides or polypeptides thatpredict the sensitivity or resistance of cells associated with a diseasestate by using said expression pattern of said microarray.
 30. Themethod according to claim 23 wherein the compounds are a member of thegroup consisting of: a.) the compounds according to claim 5; b.) thecompounds according to claim 6; c.) the compounds according to claim 7;d.) the compounds according to claim 13; e.) the compounds according toclaim 14; and f.) the compounds according to claim 15
 31. The methodaccording to claim 29 wherein said disease is colon cancer.
 32. A methodfor predicting whether an individual requiring treatment for a diseasestate, will successfully respond or will not respond to said treatmentcomprising the steps of: a.) obtaining a sample of cells from saidindividual; b.) determining whether said cells express a plurality ofmarkers; and c.) correlating the expression of said markers to theindividuals ability to respond to said treatment.
 33. The methodaccording to claim 32 wherein the plurality of markers arepolynucleotides.
 34. The method according to claim 33 wherein thepolynucleotides are selected from the group consisting of: a.) thepolynucleotides of claim 1; b.) the polynucleotides of claim 2; c.) thepolynucleotides of claim 3; and d.) the polynucleotides of claim
 4. 35.The method according to claim 34 wherein the compounds are a member ofthe group consisting of: a) the compounds according to claim 5; b.) thecompounds according to claim 6; and c.) the compounds according to claim7.
 36. The method according to claim 33 wherein the disease state iscolon cancer.
 37. The method according to claim 34 wherein the pluralityof markers are polypeptides.
 38. The method according to claim 37wherein the polypeptides are selected from the group consisting of: a.)the polypeptides of claim 9; b.) the polypeptides of claim 10; c.) thepolypeptides of claim 11; and d.) the polypeptides of claim
 12. 39. Themethod according to claim 38 wherein the compounds are a member of thegroup consisting of: a.) the compounds according to claim 5; b.) thecompounds according to claim 6; and c.) the compounds according to claim7.
 40. The method according to claim 37 wherein the disease state iscolon cancer.